Multiple exon skipping compositions for DMD

ABSTRACT

Provided are antisense molecules capable of binding to a selected target site in the human dystrophin gene to induce exon skipping, and methods of use thereof to treat muscular dystrophy.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.14/523,610, filed Oct. 24, 2014, now pending, which is a divisional ofU.S. patent application Ser. No. 12/605,276, filed Oct. 23, 2009, nowissued as U.S. Pat. No. 8,871,918, which claims the benefit of U.S.Provisional Patent Application No. 61/108,416, filed Oct. 24, 2008. Thecontents of the aforementioned applications are hereby incorporated byreference.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe sequence Listing is AVN009DVCN2_Sequence_Listing.txt. The text fileis 157 KB, was created on Sep. 10, 2015 and is being submittedelectronically via EFS-Web.

FIELD OF THE INVENTION

The present invention relates to novel antisense compounds andcompositions suitable for facilitating exon skipping in the humandystrophin gene. It also provides methods for inducing exon skippingusing the antisense compositions adapted for use in the methods of theinvention.

BACKGROUND OF THE INVENTION

Antisense technologies are being developed using a range of chemistriesto affect gene expression at a variety of different levels(transcription, splicing, stability, translation). Much of that researchhas focused on the use of antisense compounds to correct or compensatefor abnormal or disease-associated genes in a wide range of indications.Antisense molecules are able to inhibit gene expression withspecificity, and because of this, many research efforts concerningoligonucleotides as modulators of gene expression have focused oninhibiting the expression of targeted genes or the function ofcis-acting elements. The antisense oligonucleotides are typicallydirected against RNA, either the sense strand (e.g., mRNA) orminus-strand in the case of some viral RNA targets. To achieve a desiredeffect of specific gene down-regulation, the oligonucleotides generallyeither promote the decay of the targeted mRNA, block translation of themRNA or block the function of cis-acting RNA elements therebyeffectively preventing either de novo synthesis of the target protein orreplication of the viral RNA.

However, such techniques are not useful where the object is toup-regulate production of the native protein or compensate for mutationsthat induce premature termination of translation such as nonsense orframe-shifting mutations. In these cases, the defective gene transcriptshould not be subjected to targeted degradation or steric inhibition, sothe antisense oligonucleotide chemistry should not promote target mRNAdecay or block translation.

In a variety of genetic diseases, the effects of mutations on theeventual expression of a gene can be modulated through a process oftargeted exon skipping during the splicing process. The splicing processis directed by complex multi-component machinery that brings adjacentexon-intron junctions in pre-mRNA into close proximity and performscleavage of phosphodiester bonds at the ends of the introns with theirsubsequent reformation between exons that are to be spliced together.This complex and highly precise process is mediated by sequence motifsin the pre-mRNA that are relatively short semi-conserved RNA segments towhich bind the various nuclear splicing factors that are then involvedin the splicing reactions. By changing the way the splicing machineryreads or recognizes the motifs involved in pre-mRNA processing, it ispossible to create differentially spliced mRNA molecules. It has nowbeen recognized that the majority of human genes are alternativelyspliced during normal gene expression, although the mechanisms involvedhave not been identified.

In cases where a normally functional protein is prematurely terminatedbecause of mutations therein, a means for restoring some functionalprotein production through antisense technology has been shown to bepossible through intervention during the splicing processes, and that ifexons associated with disease-causing mutations can be specificallydeleted from some genes, a shortened protein product can sometimes beproduced that has similar biological properties of the native protein orhas sufficient biological activity to ameliorate the disease caused bymutations associated with the exon (Sierakowska, Sambade et al. 1996;Wilton, Lloyd et al. 1999; van Deutekom, Bremmer-Bout et al. 2001; Lu,Mann et al. 2003; Aartsma-Rus, Janson et al. 2004). Kole et al. (U.S.Pat. Nos. 5,627,274; 5,916,808; 5,976,879; and 5,665,593) disclosemethods of combating aberrant splicing using modified antisenseoligonucleotide analogs that do not promote decay of the targetedpre-mRNA. Bennett et al (U.S. Pat. No. 6,210,892) describe antisensemodulation of wild-type cellular mRNA processing also using antisenseoligonucleotide analogs that do not induce RNAse H-mediated cleavage ofthe target RNA.

The process of targeted exon skipping is likely to be particularlyuseful in long genes where there are many exons and introns, where thereis redundancy in the genetic constitution of the exons or where aprotein is able to function without one or more particular exons.Efforts to redirect gene processing for the treatment of geneticdiseases associated with truncations caused by mutations in variousgenes have focused on the use of antisense oligonucleotides that either:(1) fully or partially overlap with the elements involved in thesplicing process; or (2) bind to the pre-mRNA at a position sufficientlyclose to the element to disrupt the binding and function of the splicingfactors that would normally mediate a particular splicing reaction whichoccurs at that element.

Duchenne muscular dystrophy (DMD) is caused by a defect in theexpression of the protein dystrophin. The gene encoding the proteincontains 79 exons spread out over more than 2 million nucleotides ofDNA. Any exonic mutation that changes the reading frame of the exon, orintroduces a stop codon, or is characterized by removal of an entire outof frame exon or exons or duplications of one or more exons has thepotential to disrupt production of functional dystrophin, resulting inDMD.

A less severe form of muscular dystrophy, Becker muscular dystrophy(BMD) has been found to arise where a mutation, typically a deletion ofone or more exons, results in a correct reading frame along the entiredystrophin transcript, such that translation of mRNA into protein is notprematurely terminated. If the joining of the upstream and downstreamexons in the processing of a mutated dystrophin pre-mRNA maintains thecorrect reading frame of the gene, the result is an mRNA coding for aprotein with a short internal deletion that retains some activityresulting in a Becker phenotype.

Deletions of an exon or exons which do not alter the reading frame of adystrophin protein give rise to a BMD phenotype, whereas an exondeletion that causes a frame-shift will give rise to DMD (Monaco,Bertelson et al. 1988). In general, dystrophin mutations including pointmutations and exon deletions that change the reading frame and thusinterrupt proper protein translation result in DMD. It should also benoted that some BMD and DMD patients have exon deletions coveringmultiple exons.

Although antisense molecules may provide a tool in the treatment ofDuchenne Muscular Dystrophy (DMD), attempts to induce exon skippingusing antisense molecules have had mixed success. Successful skipping ofdystrophin exon 19 from the dystrophin pre-mRNA was achieved using avariety of antisense molecules directed at the flanking splice sites ormotifs within the exon involved in exon definition as described byErrington et al., (Errington, Mann et al. 2003).

The first example of specific and reproducible exon skipping in the mdxmouse model was reported by Wilton et al (Wilton, Lloyd et al. 1999). Bydirecting an antisense molecule to the donor splice site, exon 23skipping was induced in the dystrophin mRNA within 6 hours of treatmentof the cultured cells. Wilton et al also describe targeting the acceptorregion of the mouse dystrophin pre-mRNA with longer antisenseoligonucleotides. While the first antisense oligonucleotide directed atthe intron 23 donor splice site induced exon skipping in primarycultured myoblasts, this compound was found to be much less efficient inimmortalized cell cultures expressing higher levels of dystrophin.

Despite these efforts, there remains a need for improved antisenseoligomers targeted to multiple dystrophin exons and improved muscledelivery compositions and methods for DMD therapeutic applications.

BRIEF SUMMARY OF THE INVENTION

Embodiments of the present invention relate generally to antisensecompounds capable of binding to a selected target to induce exonskipping, and methods of use thereof to induce exon skipping. In certainembodiments, it is possible to combine two or more antisenseoligonucleotides of the present invention together to induce single ormultiple exon skipping.

In certain embodiments, it is possible to improve exon skipping of asingle or multiple exons by covalently linking together two or moreantisense oligonucleotide molecules (see, e.g., Aartsma-Rus, Janson etal. 2004).

In certain embodiments, the antisense compounds of the present inventioninduce exon skipping in the human dystrophin gene, and thereby allowmuscle cells to produce a functional dystrophin protein.

The antisense oligonucleotide compounds (also referred to herein asoligomers) of the present invention typically: (i) comprise morpholinosubunits and phosphorus-containing intersubunit linkages joining amorpholino nitrogen of one subunit to a 5′ exocyclic carbon of anadjacent subunit, (ii) contain between 10-40 nucleotide bases,preferably 20-35 bases (iii) comprise a base sequence effective tohybridize to at least 12 contiguous bases of a target sequence indystrophin pre-mRNA and induce exon skipping.

In certain embodiments, the antisense compounds of the present inventionmay comprise phosphorus-containing intersubunit linkages joining amorpholino nitrogen of one subunit to a 5′ exocyclic carbon of anadjacent subunit, in accordance with the following structure (I):

wherein:

Y₁ is —O—, —S—, —NH—, or —CH₂—;

Z is O or S;

Pj is a purine or pyrimidine base-pairing moiety effective to bind, bybase-specific hydrogen bonding, to a base in a polynucleotide; and

X is fluoro, optionally substituted alkyl, optionally substitutedalkoxy, optionally substituted thioalkoxy, amino, optionally substitutedalkylamino, or optionally substituted heterocyclyl.

In certain embodiments, the above intersubunit linkages, which areuncharged, may be interspersed with linkages that are positively chargedat physiological pH, where the total number of positively chargedlinkages is between 2 and no more than half of the total number oflinkages. For example, the positively charged linkages may have theabove structure in which X is optionally substituted 1-piperazinyl. Inother embodiments, the positively charged linkages may have the abovestructure in which X is substituted 1-piperazynyl, wherein the1-piperazynyl is substituted at the 4-position with an optionallysubstituted alkyl guanidynyl moiety.

Where the antisense compound administered is effective to target asplice site of preprocessed human dystrophin, it may have a basesequence complementary to a target region containing at least 12contiguous bases in a preprocessed messenger RNA (mRNA) human dystrophintranscript. Exemplary antisense sequences include those identified bySEQ ID NOS: 1 to 569 and 612 to 633.

In certain embodiments, an antisense sequence of the present inventionis contained within:

(a) any of the sequences identified by SEQ ID NOS: 1-20, preferably SEQID NOS: 4, 8, 11 and 12, and more preferably SEQ ID NO:12 for use inproducing skipping of exon 44 in the processing of human dystrophinpre-processed mRNA;

(b) any of the sequences identified by SEQ ID NOS: 21-76 and 612 to 624,preferably SEQ ID NOS: 27, 29, 34 and 39, and more preferably SEQ ID NO:34 for use in producing skipping of exon 45 in the processing of humandystrophin pre-processed mRNA;

(c) any of the sequences identified by SEQ ID NOS: 77-125, preferablySEQ ID NOS: 21 to 53, and more preferably SEQ ID NOS: 82, 84-87, 90 96,98, 99 and 101, for use in producing skipping of exon 46 in theprocessing of human dystrophin pre-processed mRNA;

(d) any of the sequences identified by SEQ ID NOS: 126-169, preferablySEQ ID NOS: 126-149, and more preferably SEQ ID NOS: 126, 128-130, 132,144 and 146-149, for use in producing skipping of exon 47 in theprocessing of human dystrophin pre-processed mRNA;

(e) any of the sequences identified by SEQ ID NOS: 170-224 and 634,preferably SEQ ID NOS: 170-201 and 634, and more preferably SEQ ID NOS:176, 178, 181-183, 194 and 198-201, for use in producing skipping ofexon 48 in the processing of human dystrophin pre-processed mRNA;

(f) any of the sequences identified by SEQ ID NOS: 225-266, preferablySEQ ID NOS: 225-248, and more preferably SEQ ID NOS: 227, 229, 234, 236,237 and 244-248, for use in producing skipping of exon 49 in theprocessing of human dystrophin pre-processed mRNA;

(g) any of the sequences identified by SEQ ID NOS: 267-308, preferablySEQ ID NOS: 277, 287 and 290, and more preferably SEQ ID NO: 287, foruse in producing skipping of exon 50 in the processing of humandystrophin pre-processed mRNA;

(h) any of the sequences identified by SEQ ID NOS: 309-371, preferablySEQ ID NOS: 324, 326 and 327, and more preferably SEQ ID NO: 327 for usein producing skipping of exon 51 in the processing of human dystrophinpre-processed mRNA;

(i) any of the sequences identified by SEQ ID NOS: 372-415, preferablySEQ ID NOS: 372-397, and more preferably SEQ ID NOS: 379-382, 384, 390and 392-395 for use in producing skipping of exon 52 in the processingof human dystrophin pre-processed mRNA;

(j) any of the sequences identified by SEQ ID NOS: 416-475 and 625-633,preferably SEQ ID NOS: 428, 429 and 431, and more preferably SEQ ID NO:429, for use in producing skipping of exon 53 in the processing of humandystrophin pre-processed mRNA;

(k) any of the sequences identified by SEQ ID NOS: 476-519, preferablySEQ ID NOS: 476-499, and more preferably SEQ ID NOS: 479-482, 484, 489and 491-493, for use in producing skipping of exon 54 in the processingof human dystrophin pre-processed mRNA; and

(l) any of the sequences identified by SEQ ID NOS: 520-569 and 635,preferably SEQ ID NOS: 520-546 and 635, and more preferably SEQ ID NOS:524-528, 537, 539, 540, 542 and 544, for use in producing skipping ofexon 55 in the processing of human dystrophin pre-processed mRNA;

In certain embodiments, the compound may be conjugated to anarginine-rich polypeptide effective to promote uptake of the compoundinto cells. Exemplary peptides include those identified by SEQ ID NOS:570 to 578, among others described herein.

In one exemplary embodiment, the arginine-rich polypeptide is covalentlycoupled at its N-terminal or C-terminal residue to the 3′ or 5′ end ofthe antisense compound. Also in an exemplary embodiment, the antisensecompound is composed of morpholino subunits and phosphorus-containingintersubunit linkages joining a morpholino nitrogen of one subunit to a5′ exocyclic carbon of an adjacent subunit.

In general, the peptide-oligomer conjugate may further comprise a homingpeptide which is selective for a selected mammalian tissue, i.e., thesame tissue being targeted by the cell-penetrating peptide. Theconjugate may be of the form: cell penetrating peptide-homingpeptide-antisense oligomer, or, more preferably, of the form: homingpeptide-cell penetrating peptide-antisense oligomer. For example, apeptide conjugate compound for use in treating Duchenne musculardystrophy, as described above, can further comprise a homing peptidewhich is selective for muscle tissue, such as the peptide having thesequence identified as SEQ ID NO: 579, conjugated to thecell-penetrating peptide. Exemplary conjugates of this type includethose represented herein as CP06062-MSP-PMO (cell penetratingpeptide-homing peptide-antisense oligomer) and as MSP-CP06062-PMO(homing peptide-cell penetrating peptide-antisense oligomer) (see SEQ IDNOs: 580-583).

In some embodiments, the peptide is conjugated to the oligomer via alinker moiety. In certain embodiments the linker moiety may comprise anoptionally substituted piperazynyl moiety. In other embodiments, thelinker moiety may further comprise a beta alanine and/or a6-aminohexanoic acid subunit. In yet other embodiments, the peptide isconjugated directly to the oligomer without a linker moiety.

Conjugation of the peptide to the oligomer may be at any positionsuitable for forming a covalent bond between the peptide and theoligomer or between the linker moiety and the oligomer. For example, insome embodiments conjugation of the peptide may be at the 3′ end of theoligomer. In other embodiments, conjugation of the peptide to theoligomer may be at the 5′ end of the oligomer. In yet other embodiments,the peptide may be conjugated to the oligomer through any of theintersubunit linkages.

In some embodiments, the peptide is conjugated to the oligomer at the 5′end of the oligomer. In embodiments comprising phosphorus-containingintersubunit linkages, the peptide may be conjugated to the oligomer viaa covalent bond to the phosphorous of the terminal linkage group.Conjugation in this manner may be with or without the linker moietydescribed above.

In yet other embodiments, the peptide may be conjugated to the oligomerat the 3′ end of the oligomer. In some further embodiments, the peptidemay be conjugated to the nitrogen atom of the 3′ terminal morpolinogroup of the oligomer. In this respect, the peptide may be conjugated tothe oligomer directly or via the linker moiety described above.

In some embodiments, the oligomer may be conjugated to a moiety thatenhances the solubility of the oligomer in aqueous medium. In someembodiments, the moiety that enhances solubility of the oligomer inaqueous medium is a polyethyleneglycol. In yet further embodiments, themoiety that enhances solubility of the oligomer in aqueous medium istriethylene glycol. For example, in some embodiments the moiety thatenhances solubility in aqueous medium may be conjugated to the oligomerat the 5′ end of the oligomer. Conjugation of the moiety that enhancessolubility of the oligomer in aqueous medium to the oligomer may beeither directly or through the linker moiety described above.

Certain embodiments of the present invention provide antisense moleculesselected and or adapted to aid in the prophylactic or therapeutictreatment of a genetic disorder comprising at least an antisensemolecule in a form suitable for delivery to a patient.

Certain embodiments of the invention provide methods for treating apatient suffering from a genetic disease wherein there is a mutation ina gene encoding a particular protein and the affect of the mutation canbe abrogated by exon skipping, comprising the steps of: (a) selecting anantisense molecule in accordance with the methods described herein; and(b) administering the molecule to a patient in need of such treatment.The present invention also includes the use of purified and isolatedantisense oligonucleotides of the invention, for the manufacture of amedicament for treatment of a genetic disease.

Certain embodiments provide a method of treating muscular dystrophy,such as a condition characterized by Duchenne muscular dystrophy, whichmethod comprises administering to a patient in need of treatment aneffective amount of an appropriately designed antisense oligonucleotide,as described herein, relevant to the particular genetic lesion in thatpatient. Further, certain embodiments provide a method forprophylactically treating a patient to prevent or at least minimizemuscular dystrophy, including Duchene muscular dystrophy, comprising thestep of: administering to the patient an effective amount of anantisense oligonucleotide or a pharmaceutical composition comprising oneor more of these biological molecules.

Certain embodiments relate to methods of treating muscular dystrophy ina subject, comprising administering to the subject an effective amountof a substantially uncharged antisense compound containing 20-35morpholino subunits linked by phosphorus-containing intersubunitlinkages joining a morpholino nitrogen of one subunit to a 5′ exocycliccarbon of an adjacent subunit, comprising a sequence selected from thegroup consisting SEQ ID NOS:1 to 569 and 612 to 635, and capable offorming with the complementary mRNA sequence in a dystrophin-gene exon aheteroduplex structure between said compound and mRNA having a Tm of atleast 45° C., wherein the exon is selected from the group consisting ofexons 44-55.

In certain embodiments, the muscular dystrophy is Duchenne's musculardystrophy (DMD). In certain embodiments, the muscular dystrophy isBecker muscular dystrophy (BMD).

In certain embodiments, the sequence is selected from the groupconsisting SEQ ID NOS: 1-20, and the exon is exon 44. In certainembodiments, the sequence is selected from the group consisting SEQ IDNOS: 21-76 and 612 to 624, and the exon is exon 45.

In certain embodiments, the sequence is selected from the groupconsisting SEQ ID NOS: 77-125, and the exon is exon 46. In certainembodiments, the sequence selected from the group consisting SEQ ID NOS:126-169, and the exon is exon 47.

In certain embodiments, the sequence is selected from the groupconsisting SEQ ID NOS: 170-224 and 634, and the exon is exon 48. Incertain embodiments, the sequence selected from the group consisting SEQID NOS: 225-266, and the exon is exon 49.

In certain embodiments, the sequence is selected from the groupconsisting SEQ ID NOS: 267-308, and the exon is exon 50. In certainembodiments, the sequence is selected from the group consisting SEQ IDNOS: 309-371, and the exon is exon 51.

In certain embodiments, the sequence is selected from the groupconsisting SEQ ID NOS: 372-415, and the exon is exon 52. In certainembodiments, the sequence is selected from the group consisting SEQ IDNOS: 416-475 and 625-633, and the exon is exon 53. In certainembodiments, the sequence is selected from the group consisting SEQ IDNOS: 476-519, and the exon is exon 54. In certain embodiments, thesequence is selected from the group consisting SEQ ID NOS: 520-569 and635, and the exon is exon 55. In certain embodiments, the sequencecomprises or consists essentially of SEQ ID NO:287.

Certain embodiments provide kits for treating a genetic disease, whichkits comprise at least an antisense oligonucleotide of the presentinvention, packaged in a suitable container and instructions for itsuse.

These and other objects and features will be more fully understood whenthe following detailed description of the invention is read inconjunction with the figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows an exemplary morpholino oligomer structure with aphosphorodiamidate linkage;

FIG. 1B shows a conjugate of an arginine-rich peptide and an antisenseoligomer, in accordance with an embodiment of the invention;

FIG. 1C shows a conjugate as in FIG. 1B, wherein the backbone linkagescontain one or more positively charged groups;

FIGS. 1D-G show the repeating subunit segment of exemplary morpholinooligonucleotides, designated D through G.

FIG. 2A shows the relative location and results of an antisense oligomerexon 51 scan designed to induce skipping of human dystrophin exon 51.

FIG. 2B-C shows the relative activity in cultured human rhabdomyosarcoma(RD) cells and human primary skeletal muscle cells of the three bestoligomers selected from the exon 51 scan (SEQ ID NOs: 324, 326 and 327)relative to sequences (AVI-5658; SEQ ID NO: 588 and h51AON1; SEQ IDNO:594) that are effective at inducing exon 51 skipping. FIG. 2D showsthe relative location within exon 51 of three selected oligomerscompared to certain sequences.

FIG. 3A shows the relative location and results of an antisense oligomerexon 50 scan designed to induce skipping of human dystrophin exon 50compared to other sequences that induce exon 50 skipping.

FIG. 3B shows the relative location and activity of antisense sequencesselected from the exon 50 scan (SEQ ID NOS: 277, 287, 290 and 291)compared to other sequences (SEQ ID NOS: 584 and 585).

FIG. 4A shows the relative location and results of an antisense oligomerexon 53 scan designed to induce skipping of human dystrophin exon 53.FIG. 4B shows the relative location of certain sequences used to comparethe exon-skipping activity of those oligomers selected as being mostactive in the exon 53 scan.

FIGS. 4C-F show the results of dose-ranging studies, summarized in FIG.4G, using the oligomers selected as being most efficacious in the exon53 scan (SEQ ID NOS:422, 428, 429 and 431).

FIGS. 4H and 4I show the relative activity of certain sequences (SEQ IDNOS: 608-611) compared to the activity of the most active exon53-skipping oligomer (SEQ ID NO:429) in both RD cells and human primaryskeletal muscle cells.

FIG. 5A shows the relative location and results of an antisense oligomerexon 44 scan designed to induce skipping of human dystrophin exon 44.FIG. 5B shows the relative location within exon 44 of certain sequencesused to compare the exon-skipping activity to those oligomers selectedas being most active in the exon 44 scan.

FIGS. 5C-G show the results of dose-ranging studies, summarized in FIG.5H, using the oligomers selected as being most efficacious in the exon44 scan (SEQ ID NOS: 4, 8, 11, 12 and 13).

FIGS. 5I and 5J show the relative activity of certain sequences (SEQ IDNOS: 600-603) compared to the activity of the most active exon53-skipping oligomer (SEQ ID NO:12) in both RD cells and human primaryskeletal muscle cells.

FIG. 6A shows the relative location and results of an antisense oligomerexon 45 scan designed to induce skipping of human dystrophin exon 45.FIG. 6B shows the relative location within exon 45 of certain sequencesused to compare the exon-skipping activity to those oligomers selectedas being most active in the exon 45 scan.

FIGS. 6C-F show the results of dose-ranging studies, summarized in FIG.6H, using the oligomers selected as being most efficacious in the exon45 scan (SEQ ID NOS: 27, 29, 34 and 39). FIG. 6G uses a relativelyinactive oligomer (SEQ ID NO: 49) as a negative control.

FIGS. 6I and 6J show the relative activity of certain sequences (SEQ IDNOS: 604-607) compared to the activity of the most active exon53-skipping oligomer (SEQ ID NO: 34) in both RD cells and human primaryskeletal muscle cells.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the present invention relate generally to improvedantisense compounds, and methods of use thereof, which are specificallydesigned to induce exon skipping in the dystrophin gene. Dystrophinplays a vital role in muscle function, and various muscle-relateddiseases are characterized by mutated forms of this gene. Hence, incertain embodiments, the improved antisense compounds described hereininduce exon skipping in mutated forms of the human dystrophin gene, suchas the mutated dystrophin genes found in Duchenne's muscular dystrophy(DMD) and Becker's muscular dystrophy (BMD).

Due to aberrant mRNA splicing events caused by mutations, these mutatedhuman dystrophin genes either express defective dystrophin protein orexpress no measurable dystrophin at all, a condition that leads tovarious forms of muscular dystrophy. To remedy this condition, theantisense compounds of the present invention typically hybridize toselected regions of a pre-processed RNA of a mutated human dystrophingene, induce exon skipping and differential splicing in that otherwiseaberrantly spliced dystrophin mRNA, and thereby allow muscle cells toproduce an mRNA transcript that encodes a functional dystrophin protein.In certain embodiments, the resulting dystrophin protein is notnecessarily the “wild-type” form of dystrophin, but is rather atruncated, yet functional or semi-functional, form of dystrophin.

By increasing the levels of functional dystrophin protein in musclecells, these and related embodiments may be useful in the prophylaxisand treatment of muscular dystrophy, especially those forms of musculardystrophy, such as DMD and BMD, that are characterized by the expressionof defective dystrophin proteins due to aberrant mRNA splicing. Thespecific oligomers described herein further provide improved,dystrophin-exon-specific targeting over other oligomers in use, andthereby offer significant and practical advantages over alternatemethods of treating relevant forms of muscular dystrophy.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, preferred methods andmaterials are described. For the purposes of the present invention, thefollowing terms are defined below.

DEFINITIONS

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

By “about” is meant a quantity, level, value, number, frequency,percentage, dimension, size, amount, weight or length that varies by asmuch as 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a referencequantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length.

By “coding sequence” is meant any nucleic acid sequence that contributesto the code for the polypeptide product of a gene. By contrast, the term“non-coding sequence” refers to any nucleic acid sequence that does notcontribute to the code for the polypeptide product of a gene.

Throughout this specification, unless the context requires otherwise,the words “comprise,” “comprises,” and “comprising” will be understoodto imply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements.

By “consisting of” is meant including, and limited to, whatever followsthe phrase “consisting of.” Thus, the phrase “consisting of” indicatesthat the listed elements are required or mandatory, and that no otherelements may be present.

By “consisting essentially of” is meant including any elements listedafter the phrase, and limited to other elements that do not interferewith or contribute to the activity or action specified in the disclosurefor the listed elements. Thus, the phrase “consisting essentially of”indicates that the listed elements are required or mandatory, but thatother elements are optional and may or may not be present depending uponwhether or not they materially affect the activity or action of thelisted elements.

The terms “complementary” and “complementarity” refer to polynucleotides(i.e., a sequence of nucleotides) related by the base-pairing rules. Forexample, the sequence “A-G-T,” is complementary to the sequence “T-C-A.”Complementarity may be “partial,” in which only some of the nucleicacids' bases are matched according to the base pairing rules. Or, theremay be “complete” or “total” complementarity between the nucleic acids.The degree of complementarity between nucleic acid strands hassignificant effects on the efficiency and strength of hybridizationbetween nucleic acid strands. While perfect complementarity is oftendesired, some embodiments can include one or more but preferably 6, 5,4, 3, 2, or 1 mismatches with respect to the target RNA. Variations atany location within the oligomer are included. In certain embodiments,variations in sequence near the termini of an oligomer are generallypreferable to variations in the interior, and if present are typicallywithin about 6, 5, 4, 3, 2, or 1 nucleotides of the 5′ and/or 3′terminus.

The terms “cell penetrating peptide” or “CPP” are used interchangeablyand refer to cationic cell penetrating peptides, also called transportpeptides, carrier peptides, or peptide transduction domains. Thepeptides, as shown herein, have the capability of inducing cellpenetration within 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of cellsof a given cell culture population, including all integers in between,and allow macromolecular translocation within multiple tissues in vivoupon systemic administration.

The terms “antisense oligomer” or “antisense compound” are usedinterchangeably and refer to a sequence of cyclic subunits, each bearinga base-pairing moiety, linked by intersubunit linkages that allow thebase-pairing moieties to hybridize to a target sequence in a nucleicacid (typically an RNA) by Watson-Crick base pairing, to form a nucleicacid:oligomer heteroduplex within the target sequence. The cyclicsubunits are based on ribose or another pentose sugar or, in a preferredembodiment, a morpholino group (see description of morpholino oligomersbelow).

Such an antisense oligomer can be designed to block or inhibittranslation of mRNA or to inhibit natural pre-mRNA splice processing,and may be said to be “directed to” or “targeted against” a targetsequence with which it hybridizes. In certain embodiments, the targetsequence includes a region including an AUG start codon of an mRNA, a 3′or 5′ splice site of a pre-processed mRNA, or a branch point. The targetsequence may be within an exon or within an intron. The target sequencefor a splice site may include an mRNA sequence having its 5′ end 1 toabout 25 base pairs downstream of a normal splice acceptor junction in apreprocessed mRNA. A preferred target sequence for a splice is anyregion of a preprocessed mRNA that includes a splice site or iscontained entirely within an exon coding sequence or spans a spliceacceptor or donor site. An oligomer is more generally said to be“targeted against” a biologically relevant target, such as a protein,virus, or bacteria, when it is targeted against the nucleic acid of thetarget in the manner described above. Included are antisense oligomersthat comprise, consist essentially of, or consist of one or more of SEQID NOS:1 to 569 and 612 to 635. Also included are variants of theseantisense oligomers, including variant oligomers having 80%, 85%, 90%,95%, 97%, 98%, or 99% (including all integers in between) sequenceidentity or sequence homology to any one of SEQ ID NOS:1 to 569 and 612to 635, and/or variants that differ from these sequences by about 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, preferably those variants thatinduce exon skipping of one or more selected human dystrophin exons.Also included are oligomers of any on or more of SEQ ID NOS:584-611 and634-635, which comprise a suitable number of charged linkages, asdescribed herein, e.g. up to about 1 per every 2-5 uncharged linkages,such as about 4-5 per every 10 uncharged linkages, and/or which comprisean Arg-rich peptide attached thereto, as also described herein.

The terms “morpholino oligomer” or “PMO” (phosphoramidate- orphosphorodiamidate morpholino oligomer) refer to an oligonucleotideanalog composed of morpholino subunit structures, where (i) thestructures are linked together by phosphorus-containing linkages, one tothree atoms long, preferably two atoms long, and preferably uncharged orcationic, joining the morpholino nitrogen of one subunit to a 5′exocyclic carbon of an adjacent subunit, and (ii) each morpholino ringbears a purine or pyrimidine base-pairing moiety effective to bind, bybase specific hydrogen bonding, to a base in a polynucleotide. See,e.g., the structure in FIG. 1A, which shows a preferredphosphorodiamidate linkage type. Variations can be made to this linkageas long as they do not interfere with binding or activity. For example,the oxygen attached to phosphorus may be substituted with sulfur(thiophosphorodiamidate). The 5′ oxygen may be substituted with amino orlower alkyl substituted amino. The pendant nitrogen attached tophosphorus may be unsubstituted, monosubstituted, or disubstituted with(optionally substituted) lower alkyl. See also the discussion ofcationic linkages below. The synthesis, structures, and bindingcharacteristics of morpholino oligomers are detailed in U.S. Pat. Nos.5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, and5,506,337, and PCT Appn. No. PCT/US07/11435 (cationic linkages), all ofwhich are incorporated herein by reference.

The purine or pyrimidine base pairing moiety is typically adenine,cytosine, guanine, uracil, thymine or inosine. Also included are basessuch as pyridin-4-one, pyridin-2-one, phenyl, pseudouracil,2,4,6-trime115thoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl,aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines(e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine), propyne,quesosine, 2-thiouridine, 4-thiouridine, wybutosine, wybutoxosine,4-acetyltidine, 5-(carboxyhydroxymethyl)uridine,5′-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluridine, β-D-galactosylqueosine,1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine,3-methylcytidine, 2-methyladenosine, 2-methylguanosine,N6-methyladenosine, 7-methylguanosine,5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine,5-methylcarbonyhnethyluridine, 5-methyloxyuridine,5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine,β-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine,threonine derivatives and others (Burgin et al., 1996, Biochemistry, 35,14090; Uhlman & Peyman, supra). By “modified bases” in this aspect ismeant nucleotide bases other than adenine (A), guanine (G), cytosine(C), thymine (T), and uracil (U), as illustrated above; such bases canbe used at any position in the antisense molecule. Persons skilled inthe art will appreciate that depending on the uses of the oligomers, Tsand Us are interchangeable. For instance, with other antisensechemistries such as 2′-O-methyl antisense oligonucleotides that are moreRNA-like, the T bases may be shown as U (see, e.g., Sequence IDListing).

An “amino acid subunit” or “amino acid residue” can refer to an α-aminoacid residue (e.g., —CO—CHR—NH—) or a β- or other amino acid residue(e.g., —CO—(CH₂)_(n)CHR—NH—), where R is a side chain (which may includehydrogen) and n is 1 to 6, preferably 1 to 4.

The term “naturally occurring amino acid” refers to an amino acidpresent in proteins found in nature, such as the 20 (L)-amino acidsutilized during protein biosynthesis as well as others such as4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine,citrulline and ornithine. The term “non-natural amino acids” refers tothose amino acids not present in proteins found in nature, examplesinclude beta-alanine (β-Ala; or B), 6-aminohexanoic acid (Ahx) and6-aminopentanoic acid. Additional examples of “non-natural amino acids”include, without limitation, (D)-amino acids, norleucine, norvaline,p-fluorophenylalanine, ethionine and the like, which are known to aperson skilled in the art.

An “effective amount” or “therapeutically effective amount” refers to anamount of therapeutic compound, such as an antisense oligomer,administered to a mammalian subject, either as a single dose or as partof a series of doses, which is effective to produce a desiredphysiological response or therapeutic effect in the subject. One exampleof a desired physiological response includes increased expression of arelatively functional or biologically active form of the dystrophinprotein, mainly in muscle tissues or cells that contain a defectivedystrophin protein or no dystrophin, as compared no antisense oligomeror a control oligomer. Examples of desired therapeutic effects include,without limitation, improvements in the symptoms or pathology ofmuscular dystrophy, reducing the progression of symptoms or pathology ofmuscular dystrophy, and slowing the onset of symptoms or pathology ofmuscular dystrophy, among others. Examples of such symptoms includefatigue, mental retardation, muscle weakness, difficulty with motorskills (e.g., running, hopping, jumping), frequent falls, and difficultywalking. The pathology of muscular dystrophy can be characterized, forexample, by muscle fibre damage and membrane leakage. For an antisenseoligomer, this effect is typically brought about by altering thesplice-processing of a selected target sequence (e.g., dystrophin), suchas to induce exon skipping.

An “exon” refers to a defined section of nucleic acid that encodes for aprotein, or a nucleic acid sequence that is represented in the matureform of an RNA molecule after either portions of a pre-processed (orprecursor) RNA have been removed by splicing. The mature RNA moleculecan be a messenger RNA (mRNA) or a functional form of a non-coding RNA,such as rRNA or tRNA. The human dystrophin gene has about 75 exons.

An “intron” refers to a nucleic acid region (within a gene) that is nottranslated into a protein. An intron is a non-coding section that istranscribed into a precursor mRNA (pre-mRNA), and subsequently removedby splicing during formation of the mature RNA.

“Exon skipping” refers generally to the process by which an entire exon,or a portion thereof, is removed from a given pre-processed RNA, and isthereby excluded from being present in the mature RNA, such as themature mRNA that is translated into a protein. Hence, the portion of theprotein that is otherwise encoded by the skipped exon is not present inthe expressed form of the protein, typically creating an altered, thoughstill functional, form of the protein. In certain embodiments, the exonbeing skipped is an aberrant exon from the human dystrophin gene, whichmay contain a mutation or other alteration in its sequence thatotherwise causes aberrant splicing. In certain embodiments, the exonbeing skipped is any one or more of exons 1-75 of the dystrophin gene,though any one or more of exons 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, and/or 55 of the human dystrophin gene are preferred.

“Dystrophin” is a rod-shaped cytoplasmic protein, and a vital part ofthe protein complex that connects the cytoskeleton of a muscle fiber tothe surrounding extracellular matrix through the cell membrane.Dystrophin contains multiple functional domains. For instance,dystrophin contains an actin binding domain at about amino acids 14-240and a central rod domain at about amino acids 253-3040. This largecentral domain is formed by 24 spectrin-like triple-helical elements ofabout 109 amino acids, which have homology to alpha-actinin andspectrin. The repeats are typically interrupted by four proline-richnon-repeat segments, also referred to as hinge regions. Repeats 15 and16 are separated by an 18 amino acid stretch that appears to provide amajor site for proteolytic cleavage of dystrophin. The sequence identitybetween most repeats ranges from 10-25%. One repeat contains threealpha-helices: 1, 2 and 3. Alpha-helices 1 and 3 are each formed by 7helix turns, probably interacting as a coiled-coil through a hydrophobicinterface. Alpha-helix 2 has a more complex structure and is formed bysegments of four and three helix turns, separated by a Glycine orProline residue. Each repeat is encoded by two exons, typicallyinterrupted by an intron between amino acids 47 and 48 in the first partof alpha-helix 2. The other intron is found at different positions inthe repeat, usually scattered over helix-3. Dystrophin also contains acysteine-rich domain at about amino acids 3080-3360), including acysteine-rich segment (i.e., 15 Cysteines in 280 amino acids) showinghomology to the C-terminal domain of the slime mold (Dictyosteliumdiscoideum) alpha-actinin. The carboxy-terminal domain is at about aminoacids 3361-3685.

The amino-terminus of dystrophin binds to F-actin and thecarboxy-terminus binds to the dystrophin-associated protein complex(DAPC) at the sarcolemma. The DAPC includes the dystroglycans,sarcoglycans, integrins and caveolin, and mutations in any of thesecomponents cause autosomally inherited muscular dystrophies. The DAPC isdestabilized when dystrophin is absent, which results in diminishedlevels of the member proteins, and in turn leads to progressive fibredamage and membrane leakage. In various forms of muscular dystrophy,such as Duchenne's muscular dystrophy (DMD) and Becker's musculardystrophy (BMD), muscle cells produce an altered and functionallydefective form of dystrophin, or no dystrophin at all, mainly due tomutations in the gene sequence that lead to incorrect splicing. Thepredominant expression of the defective dystrophin protein, or thecomplete lack of dystrophin or a dystrophin-like protein, leads to rapidprogression of muscle degeneration, as noted above. In this regard, a“defective” dystrophin protein may be characterized by the forms ofdystrophin that are produced in certain subjects with DMD or BMD, asknown in the art, or by the absence of detectable dystrophin.

Table A provides an illustration of the various dystrophin domains, theamino acid residues that encompass these domains, and the exons thatencode them.

TABLE A Residue Domain Sub Domain Nos Exons actin binding  14-240 2-8domain central rod  253-3040  8-61 domain hinge 1 253-327 (8)-9   repeat1 337-447 10-11 repeat 2 448-556 12-14 repeat 3 557-667 14-16 hinge 2668-717 17 repeat 4 718-828 (17)-20   repeat 5 829-934 20-21 repeat 6 935-1045 22-23 repeat 7 1046-1154 (23)-(26) repeat 8 1155-1263 26-27repeat 9 1264-1367   28-(30) repeat 10 1368-1463 30-32 repeat 111464-1568   32-(34) repeat 12 1569-1676 34-35 repeat 13 1677-1778 36-37repeat 14 1779-1874   38-(40) repeat 15 1875-1973 40-41 interruption1974-1991 42 repeat 16 1992-2101 42-43 repeat 17 2102-2208 44-45 repeat18 2209-2318 46-48 repeat 19 2319-2423 48-50 hinge 3 2424-2470 50-51repeat 20 2471-2577 51-53 repeat 21 2578-2686   53-(55) repeat 222687-2802   55-(57) repeat 23 2803-2931 57-59 repeat 24 2932-3040  59-(61) hinge 4 3041-3112 61-64 Cysteine-rich 3080-3360 63-69 domaindystroglycan binding site 3080-3408 63-70 WW domain 3056-3092 62-63EF-hand 1 3130-3157 65 EF-hand 2 3178-3206 65-66 ZZ domain 3307-335468-69 Carboxy-terminal 3361-3685 70-79 domain alpha1-syntrophin binding3444-3494 73-74 site β1-syntrophin binding site 3495-3535 74-75(Leu)6-heptad repeat 3558-3593 75

As used herein, the terms “function” and “functional” and the like referto a biological, enzymatic, or therapeutic function.

A “functional” dystrophin protein refers generally to a dystrophinprotein having sufficient biological activity to reduce the progressivedegradation of muscle tissue that is otherwise characteristic ofmuscular dystrophy, typically as compared to the altered or “defective”form of dystrophin protein that is present in certain subjects with DMDor BMD. In certain embodiments, a functional dystrophin protein may haveabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (includingall integers in between) of the in vitro or in vivo biological activityof wild-type dystrophin, as measured according to routine techniques inthe art. As one example, dystrophin-related activity in muscle culturesin vitro can be measured according to myotube size, myofibrilorganization (or disorganization), contractile activity, and spontaneousclustering of acetylcholine receptors (see, e.g., Brown et al., Journalof Cell Science. 112:209-216, 1999). Animal models are also valuableresources for studying the pathogenesis of disease, and provide a meansto test dystrophin-related activity. Two of the most widely used animalmodels for DMD research are the mdx mouse and the golden retrievermuscular dystrophy (GRMD) dog, both of which are dystrophin negative(see, e.g., Collins & Morgan, Int J Exp Pathol 84: 165-172, 2003). Theseand other animal models can be used to measure the functional activityof various dystrophin proteins. Included are truncated forms ofdystrophin, such as those forms that are produced by certain of theexon-skipping antisense compounds of the present invention.

By “gene” is meant a unit of inheritance that occupies a specific locuson a chromosome and consists of transcriptional and/or translationalregulatory sequences and/or a coding region and/or non-translatedsequences (i.e., introns, 5′ and 3′ untranslated sequences).

By “isolated” is meant material that is substantially or essentiallyfree from components that normally accompany it in its native state. Forexample, an “isolated polynucleotide,” as used herein, may refer to apolynucleotide that has been purified or removed from the sequences thatflank it in a naturally-occurring state, e.g., a DNA fragment that hasbeen removed from the sequences that are normally adjacent to thefragment.

By “enhance” or “enhancing,” or “increase” or “increasing,” or“stimulate” or “stimulating,” refers generally to the ability of one orantisense compounds or compositions to produce or cause a greaterphysiological response (i.e., downstream effects) in a cell or asubject, as compared to the response caused by either no antisensecompound or a control compound. A measurable physiological response mayinclude increased expression of a functional form of a dystrophinprotein, or increased dystrophin-related biological activity in muscletissue, among other responses apparent from the understanding in the artand the description herein. Increased muscle function can also bemeasured, including increases or improvements in muscle function byabout 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 100%. The percentage of muscle fibresthat express a functional dystrophin can also be measured, includingincreased dystrophin expression in about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%of muscle fibres. For instance, it has been shown that around 40% ofmuscle function improvement can occur if 25-30% of fibers expressdystrophin (see, e.g., DelloRusso et al, Proc Natl Acad Sci USA 99:12979-12984, 2002). An “increased” or “enhanced” amount is typically a“statistically significant” amount, and may include an increase that is1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 or more times(e.g., 500, 1000 times) (including all integers and decimal points inbetween and above 1), e.g., 1.5, 1.6, 1.7. 1.8, etc.) the amountproduced by no antisense compound (the absence of an agent) or a controlcompound.

The term “reduce” or “inhibit” may relate generally to the ability ofone or more antisense compounds of the invention to “decrease” arelevant physiological or cellular response, such as a symptom of adisease or condition described herein, as measured according to routinetechniques in the diagnostic art. Relevant physiological or cellularresponses (in vivo or in vitro) will be apparent to persons skilled inthe art, and may include reductions in the symptoms or pathology ofmuscular dystrophy, or reductions in the expression of defective formsof dystrophin, such as the altered forms of dystrophin that areexpressed in individuals with DMD or BMD. A “decrease” in a response maybe statistically significant as compared to the response produced by noantisense compound or a control composition, and may include a 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100% decrease, including all integers in between.

“Homology” refers to the percentage number of amino acids that areidentical or constitute conservative substitutions. Homology may bedetermined using sequence comparison programs such as GAP (Deveraux etal., 1984, Nucleic Acids Research 12, 387-395). In this way sequences ofa similar or substantially different length to those cited herein couldbe compared by insertion of gaps into the alignment, such gaps beingdetermined, for example, by the comparison algorithm used by GAP.

The recitations “sequence identity” or, for example, comprising a“sequence 50% identical to,” as used herein, refer to the extent thatsequences are identical on a nucleotide-by-nucleotide basis or an aminoacid-by-amino acid basis over a window of comparison. Thus, a“percentage of sequence identity” may be calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser,Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn,Gln, Cys and Met) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity.

Terms used to describe sequence relationships between two or morepolynucleotides or polypeptides include “reference sequence,”“comparison window,” “sequence identity,” “percentage of sequenceidentity,” and “substantial identity”. A “reference sequence” is atleast 8 or 10 but frequently 15 to 18 and often at least 25 monomerunits, inclusive of nucleotides and amino acid residues, in length.Because two polynucleotides may each comprise (1) a sequence (i.e., onlya portion of the complete polynucleotide sequence) that is similarbetween the two polynucleotides, and (2) a sequence that is divergentbetween the two polynucleotides, sequence comparisons between two (ormore) polynucleotides are typically performed by comparing sequences ofthe two polynucleotides over a “comparison window” to identify andcompare local regions of sequence similarity. A “comparison window”refers to a conceptual segment of at least 6 contiguous positions,usually about 50 to about 100, more usually about 100 to about 150 inwhich a sequence is compared to a reference sequence of the same numberof contiguous positions after the two sequences are optimally aligned.The comparison window may comprise additions or deletions (i.e., gaps)of about 20% or less as compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. Optimal alignment of sequences for aligning a comparisonwindow may be conducted by computerized implementations of algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage Release 7.0, Genetics Computer Group, 575 Science Drive Madison,Wis., USA) or by inspection and the best alignment (i.e., resulting inthe highest percentage homology over the comparison window) generated byany of the various methods selected. Reference also may be made to theBLAST family of programs as for example disclosed by Altschul et al.,1997, Nucl. Acids Res. 25:3389. A detailed discussion of sequenceanalysis can be found in Unit 19.3 of Ausubel et al., “Current Protocolsin Molecular Biology,” John Wiley & Sons Inc, 1994-1998, Chapter 15.

“Treatment” or “treating” of an individual (e.g., a mammal, such as ahuman) or a cell may include any type of intervention used in an attemptto alter the natural course of the individual or cell. Treatmentincludes, but is not limited to, administration of a pharmaceuticalcomposition, and may be performed either prophylactically or subsequentto the initiation of a pathologic event or contact with an etiologicagent. Treatment includes any desirable effect on the symptoms orpathology of a disease or condition associated with the dystrophinprotein, as in certain forms of muscular dystrophy, and may include, forexample, minimal changes or improvements in one or more measurablemarkers of the disease or condition being treated. Also included are“prophylactic” treatments, which can be directed to reducing the rate ofprogression of the disease or condition being treated, delaying theonset of that disease or condition, or reducing the severity of itsonset. “Treatment” or “prophylaxis” does not necessarily indicatecomplete eradication, cure, or prevention of the disease or condition,or associated symptoms thereof.

Hence, included are methods of treating muscular dystrophy, such as DMDand BMD, by administering one or more antisense oligomers of the presentinvention (e.g., SEQ ID NOS: 1 to 569 and 612 to 635, and variantsthereof), optionally as part of a pharmaceutical formulation or dosageform, to a subject in need thereof. Also included are methods ofinducing exon-skipping in a subject by administering one or moreantisense oligomers, in which the exon is one of exons 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, and/or 55 from the dystrophin gene,preferably the human dystrophin gene. A “subject,” as used herein,includes any animal that exhibits a symptom, or is at risk forexhibiting a symptom, which can be treated with an antisense compound ofthe invention, such as a subject that has or is at risk for having DMDor BMD, or any of the symptoms associated with these conditions (e.g.,muscle fibre loss). Suitable subjects (patients) include laboratoryanimals (such as mouse, rat, rabbit, or guinea pig), farm animals, anddomestic animals or pets (such as a cat or dog). Non-human primates and,preferably, human patients, are included.

Also included are vector delivery systems that are capable of expressingthe oligomeric, dystrophin-targeting sequences of the present invention,such as vectors that express a polynucleotide sequence comprising anyone or more of SEQ ID NOS: 1 to 569 and 612 to 635, or variants thereof,as described herein. By “vector” or “nucleic acid construct” is meant apolynucleotide molecule, preferably a DNA molecule derived, for example,from a plasmid, bacteriophage, yeast or virus, into which apolynucleotide can be inserted or cloned. A vector preferably containsone or more unique restriction sites and can be capable of autonomousreplication in a defined host cell including a target cell or tissue ora progenitor cell or tissue thereof, or be integrable with the genome ofthe defined host such that the cloned sequence is reproducible.Accordingly, the vector can be an autonomously replicating vector, i.e.,a vector that exists as an extra-chromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g., a linear orclosed circular plasmid, an extra-chromosomal element, amini-chromosome, or an artificial chromosome. The vector can contain anymeans for assuring self-replication. Alternatively, the vector can beone which, when introduced into the host cell, is integrated into thegenome and replicated together with the chromosome(s) into which it hasbeen integrated.

A vector or nucleic acid construct system can comprise a single vectoror plasmid, two or more vectors or plasmids, which together contain thetotal DNA to be introduced into the genome of the host cell, or atransposon. The choice of the vector will typically depend on thecompatibility of the vector with the host cell into which the vector isto be introduced. In the present case, the vector or nucleic acidconstruct is preferably one which is operably functional in a mammaliancell, such as a muscle cell. The vector can also include a selectionmarker such as an antibiotic or drug resistance gene, or a reporter gene(i.e., green fluorescent protein, luciferase), that can be used forselection or identification of suitable transformants or transfectants.Exemplary delivery systems may include viral vector systems (i.e.,viral-mediated transduction) including, but not limited to, retroviral(e.g., lentiviral) vectors, adenoviral vectors, adeno-associated viralvectors, and herpes viral vectors, among others known in the art.

The term “operably linked” as used herein means placing anoligomer-encoding sequence under the regulatory control of a promoter,which then controls the transcription of the oligomer.

A wild-type gene or gene product is that which is most frequentlyobserved in a population and is thus arbitrarily designed the “normal”or “wild-type” form of the gene.

“Alkyl” or “alkylene” both refer to a saturated straight or branchedchain hydrocarbon radical containing from 1 to 18 carbons. Examplesinclude without limitation methyl, ethyl, propyl, iso-propyl, butyl,iso-butyl, tert-butyl, n-pentyl and n-hexyl. The term “lower alkyl”refers to an alkyl group, as defined herein, containing between 1 and 8carbons.

“Alkenyl” refers to an unsaturated straight or branched chainhydrocarbon radical containing from 2 to 18 carbons and comprising atleast one carbon to carbon double bond. Examples include withoutlimitation ethenyl, propenyl, iso-propenyl, butenyl, iso-butenyl,tert-butenyl, n-pentenyl and n-hexenyl. The term “lower alkenyl” refersto an alkenyl group, as defined herein, containing between 2 and 8carbons.

“Alkynyl” refers to an unsaturated straight or branched chainhydrocarbon radical containing from 2 to 18 carbons comprising at leastone carbon to carbon triple bond. Examples include without limitationethynyl, propynyl, iso-propynyl, butynyl, iso-butynyl, tert-butynyl,pentynyl and hexynyl. The term “lower alkynyl” refers to an alkynylgroup, as defined herein, containing between 2 and 8 carbons.

“Cycloalkyl” refers to a mono- or poly-cyclic alkyl radical. Examplesinclude without limitation cyclobutyl, cycopentyl, cyclohexyl,cycloheptyl and cyclooctyl.

“Aryl” refers to a cyclic aromatic hydrocarbon moiety containing from 5to 18 carbons having one or more closed ring(s). Examples includewithout limitation phenyl, benzyl, naphthyl, anthracenyl,phenanthracenyl and biphenyl.

“Aralkyl” refers to a radical of the formula RaRb where Ra is analkylene chain as defined above and Rb is one or more aryl radicals asdefined above, for example, benzyl, diphenylmethyl and the like.

“Thioalkoxy” refers to a radical of the formula —SRc where Rc is analkyl radical as defined herein. The term “lower thioalkoxy” refers toan alkoxy group, as defined herein, containing between 1 and 8 carbons.

“Alkoxy” refers to a radical of the formula —ORda where Rd is an alkylradical as defined herein. The term “lower alkoxy” refers to an alkoxygroup, as defined herein, containing between 1 and 8 carbons. Examplesof alkoxy groups include, without limitation, methoxy and ethoxy.

“Alkoxyalkyl” refers to an alkyl group substituted with an alkoxy group.

“Carbonyl” refers to the —C(═O)— radical.

“Guanidynyl” refers to the H₂N(C═NH₂)—NH— radical.

“Amidinyl” refers to the H₂N(C═NH₂)CH— radical.

“Amino” refers to the —NH₂ radical.

“Alkylamino” refers to a radical of the formula —NHRd or —NRdRd whereeach Rd is, independently, an alkyl radical as defined herein. The term“lower alkylamino” refers to an alkylamino group, as defined herein,containing between 1 and 8 carbons.

“Heterocycle” means a 5- to 7-membered monocyclic, or 7- to 10-memberedbicyclic, heterocyclic ring which is either saturated, unsaturated, oraromatic, and which contains from 1 to 4 heteroatoms independentlyselected from nitrogen, oxygen and sulfur, and wherein the nitrogen andsulfur heteroatoms may be optionally oxidized, and the nitrogenheteroatom may be optionally quaternized, including bicyclic rings inwhich any of the above heterocycles are fused to a benzene ring. Theheterocycle may be attached via any heteroatom or carbon atom.Heterocycles include heteroaryls as defined below. Thus, in addition tothe heteroaryls listed below, heterocycles also include morpholinyl,pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl, hydantoinyl,valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,tetrahydropyranyl, tetrahydropyridinyl, tetrahydrothiophenyl,tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiopyranyl, andthe like.

“Heteroaryl” means an aromatic heterocycle ring of 5- to 10 members andhaving at least one heteroatom selected from nitrogen, oxygen andsulfur, and containing at least 1 carbon atom, including both mono- andbicyclic ring systems. Representative heteroaryls are pyridyl, furyl,benzofuranyl, thiophenyl, benzothiophenyl, quinolinyl, pyrrolyl,indolyl, oxazolyl, benzoxazolyl, imidazolyl, benzimidazolyl, thiazolyl,benzothiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, pyridazinyl,pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl, phthalazinyl, andquinazolinyl.

The terms “optionally substituted alkyl”, “optionally substitutedalkenyl”, “optionally substituted alkoxy”, “optionally substitutedthioalkoxy”, “optionally substituted alkyl amino”, “optionallysubstituted lower alkyl”, “optionally substituted lower alkenyl”,“optionally substituted lower alkoxy”, “optionally substituted lowerthioalkoxy”, “optionally substituted lower alkyl amino” and “optionallysubstituted heterocyclyl” mean that, when substituted, at least onehydrogen atom is replaced with a substituent. In the case of an oxosubstituent (═O) two hydrogen atoms are replaced. In this regard,substituents include: deuterium, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, optionally substituted heterocycle,optionally substituted cycloalkyl, oxo, halogen, —CN, —ORx, NRxRy,NRxC(═O)Ry, NRxSO2Ry, —NRxC(═O)NRxRy, C(═O)Rx, C(═O)ORx, C(═O)NRxRy,—SOmRx and —SOmNRxRy, wherein m is 0, 1 or 2, Rx and Ry are the same ordifferent and independently hydrogen, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, optionally substituted heterocycle oroptionally substituted cycloalkyl and each of said optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted aryl, optionally substitutedheterocycle and optionally substituted cycloalkyl substituents may befurther substituted with one or more of oxo, halogen, —CN, —ORx, NRxRy,NRxC(═O)Ry, NRxSO2Ry, —NRxC(═O)NRxRy, C(═O)Rx, C(═O)ORx, C(═O)NRxRy,—SOmRx and —SOmNRxRy.

Constructing Antisense Oligonucleotides

Examples of morpholino oligonucleotides having phosphorus-containingbackbone linkages are illustrated in FIGS. 1A-1C. Especially preferredis a phosphorodiamidate-linked morpholino oligonucleotide such as shownin FIG. 1C, which is modified, in accordance with one aspect of thepresent invention, to contain positively charged groups at preferably10%-50% of its backbone linkages. Morpholino oligonucleotides withuncharged backbone linkages and their preparation, including antisenseoligonucleotides, are detailed, for example, in (Summerton and Weller1997) and in co-owned U.S. Pat. Nos. 5,698,685, 5,217,866, 5,142,047,5,034,506, 5,166,315, 5,185, 444, 5,521,063, and 5,506,337, all of whichare expressly incorporated by reference herein.

Important properties of the morpholino-based subunits include: 1) theability to be linked in a oligomeric form by stable, uncharged orpositively charged backbone linkages; 2) the ability to support anucleotide base (e.g. adenine, cytosine, guanine, thymidine, uracil andinosine) such that the polymer formed can hybridize with acomplementary-base target nucleic acid, including target RNA, Tm valuesabove about 45° C. in relatively short oligonucleotides (e.g., 10-15bases); 3) the ability of the oligonucleotide to be actively orpassively transported into mammalian cells; and 4) the ability of theantisense oligonucleotide:RNA heteroduplex to resist RNAse and RNaseHdegradation, respectively.

Exemplary backbone structures for antisense oligonucleotides of theclaimed subject matter include the morpholino subunit types shown inFIGS. 1D-G, each linked by an uncharged or positively charged,phosphorus-containing subunit linkage. FIG. 1D shows aphosphorus-containing linkage which forms the five atom repeating-unitbackbone, wherein the morpholino rings are linked by a 1-atomphosphoamide linkage. FIG. 1E shows a linkage which produces a 6-atomrepeating-unit backbone. In this structure, the atom Y linking the 5′morpholino carbon to the phosphorus group may be sulfur, nitrogen,carbon or, preferably, oxygen. The X moiety pendant from the phosphorusmay be fluorine, an alkyl or substituted alkyl, an alkoxy or substitutedalkoxy, a thioalkoxy or substituted thioalkoxy, or unsubstituted,monosubstituted, or disubstituted nitrogen, including cyclic structures,such as morpholines or piperidines. Alkyl, alkoxy and thioalkoxypreferably include 1-6 carbon atoms. The Z moieties are sulfur oroxygen, and are preferably oxygen.

The linkages shown in FIGS. 1F and 1G are designed for 7-atomunit-length backbones. In structure 1F, the X moiety is as in Structure1E, and the Y moiety may be methylene, sulfur, or, preferably, oxygen.In Structure 1G, the X and Y moieties are as in Structure 1E.Particularly preferred morpholino oligonucleotides include thosecomposed of morpholino subunit structures of the form shown in FIG. 1E,where X═NH₂, N(CH₃)₂, optionally substituted 1-piperazinyl, or othercharged group, Y═O, and Z═O.

As noted above, the uncharged or substantially uncharged oligonucleotidemay be modified, in accordance with an aspect of the invention, toinclude charged linkages, e.g. up to about 1 per every 2-5 unchargedlinkages, such as about 4-5 per every 10 uncharged linkages. Optimalimprovement in antisense activity may be seen when about 25% of thebackbone linkages are cationic, including about 20% to about 30%. Alsoincluded are oligomers in which about 35%, 40%, 45%, 50%, 55%, 60%(including all integers in between), or more of the backbone linkagesare cationic. Enhancement is also seen with a small number, e.g., 5% or10-20%, of cationic linkages.

A substantially uncharged, phosphorus containing backbone in anoligonucleotide analog is typically one in which a majority of thesubunit linkages, e.g., between 50%-100%, typically at least 60% to 100%or 75% or 80% of its linkages, are uncharged at physiological pH andcontain a single phosphorous atom.

Additional experiments conducted in support of the present inventionindicate that the enhancement seen with added cationic backbone chargesmay, in some cases, be further enhanced by distributing the bulk of thecharges close to the “center-region” backbone linkages of the antisenseoligonucleotide, e.g., in a 20mer oligonucleotide with 8 cationicbackbone linkages, having at least 70% of these charged linkageslocalized in the 10 centermost linkages.

The antisense compounds can be prepared by stepwise solid-phasesynthesis, employing methods detailed in the references cited above, andbelow with respect to the synthesis of oligonucleotides having a mixtureof uncharged and cationic backbone linkages. In some cases, it may bedesirable to add additional chemical moieties to the antisense compound,e.g. to enhance pharmacokinetics or to facilitate capture or detectionof the compound. Such a moiety may be covalently attached, typically toa terminus of the oligomer, according to standard synthetic methods. Forexample, addition of a polyethyleneglycol moiety or other hydrophilicpolymer, e.g., one having 10-100 monomeric subunits, may be useful inenhancing solubility. One or more charged groups, e.g., anionic chargedgroups such as an organic acid, may enhance cell uptake. A reportermoiety, such as fluorescein or a radiolabeled group, may be attached forpurposes of detection. Alternatively, the reporter label attached to theoligomer may be a ligand, such as an antigen or biotin, capable ofbinding a labeled antibody or streptavidin. In selecting a moiety forattachment or modification of an antisense compound, it is generally ofcourse desirable to select chemical compounds of groups that arebiocompatible and likely to be tolerated by a subject withoutundesirable side effects.

As noted above, the antisense compound can be constructed to contain aselected number of cationic linkages interspersed with unchargedlinkages of the type described above. The intersubunit linkages, bothuncharged and cationic, preferably are phosphorus-containing linkages,having the structure (II):

wherein:

W is —S— or —O—, and is preferably —O—,

X=—NR¹R² or —OR⁶,

Y=—O— or —NR′, and

each said linkage in the oligomer is selected from:

(a) an uncharged linkage (a), wherein each of R¹, R², R⁶ and R⁷ isindependently selected from hydrogen and lower alkyl;

(b1) a cationic linkage (b1), wherein X=—NR¹R² and Y=—O—, and —NR¹R²represents an optionally substituted piperazinyl moiety, such thatR¹R²=—CHRCHRN(R³)(R⁴)CHRCHR—, wherein:

each R is independently H or —CH₃,

R⁴ is H, —CH₃, or an electron pair, and

R³ is selected from H, optionally substituted lower alkyl, —C(═NH)NH₂,—Z-L-NHC(═NH)NH₂, and [—C(═O)CHR′NH]_(m)H, where: Z is —C(═O)— or adirect bond, L is an optional linker up to 18 atoms in length,preferably up to 12 atoms, and more preferably up to 8 atoms in length,having bonds selected from optionally substituted alkyl, optionallysubstituted alkoxy, and optionally substituted alkylamino, R′ is a sidechain of a naturally occurring amino acid or a one- or two-carbonhomolog thereof, and m is 1 to 6, preferably 1 to 4;

(b2) a cationic linkage (b2), wherein X=—NR¹R² and Y=—O—, R¹=H or —CH₃,and R²=LNR³R⁴R⁵, wherein L, R³, and R⁴ are as defined above, and R⁵ isH, optionally substituted lower alkyl, or optionally substituted lower(alkoxy)alkyl; and

(b3) a cationic linkage (b3), wherein Y=—NR′ and X=—OR⁶, andR⁷=-LNR³R⁴R⁵, wherein L, R³, R⁴ and R⁵ are as defined above, and R⁶ is Hor optionally substituted lower alkyl; and at least one said linkage isselected from cationic linkages (b1), (b2), and (b3).

Preferably, the oligomer includes at least two consecutive linkages oftype (a) (i.e. uncharged linkages). In further embodiments, at least 5%of the linkages in the oligomer are cationic linkages (i.e. type (b1),(b2), or (b3)); for example, 10% to 60%, and preferably 20-50% linkagesmay be cationic linkages.

In one embodiment, at least one linkage is of type (b1), where,preferably, each R is H, R⁴ is H, —CH₃, or an electron pair, and R³ isselected from H, optionally substituted lower alkyl, —C(═NH)NH₂, and—C(═O)-L-NHC(═NH)NH₂. The latter two embodiments of R³ provide aguanidino moiety, either attached directly to the piperazine ring, orpendant to a linker group L, respectively. For ease of synthesis, thevariable Z in R³ is preferably —C(═O)—, as shown.

The linker group L, as noted above, contains bonds in its backboneselected from optionally substituted alkyl, optionally substitutedalkoxy, and optionally substituted alkylamino, wherein the terminalatoms in L (e.g., those adjacent to carbonyl or nitrogen) are carbonatoms. Although branched linkages are possible, the linker is preferablyunbranched. In one embodiment, the linker is a linear alkyl linker. Sucha linker may have the structure —(CH₂)_(n)—, where n is 1-12, preferably2-8, and more preferably 2-6.

The morpholino subunits have the following structure (III):

wherein Pi is a base-pairing moiety, and the linkages depicted aboveconnect the nitrogen atom of (III) to the 5′ carbon of an adjacentsubunit. The base-pairing moieties Pi may be the same or different, andare generally designed to provide a sequence which binds to a targetnucleic acid.

The use of embodiments of linkage types (b1), (b2) and (b3) above tolink morpholino subunits (III) may be illustrated graphically asfollows:

Preferably, all cationic linkages in the oligomer are of the same type;i.e. all of type (b1), all of type (b2), or all of type (b3).

In further embodiments, the cationic linkages are selected from linkages(b1′) and (b1″) as shown below, where (b1′) is referred to herein as a“Pip” linkage and (b1″) is referred to herein as a “GuX” linkage:

In the structures above, W is S or O, and is preferably O; each of R¹and R² is independently selected from hydrogen and optionallysubstituted lower alkyl, and is preferably methyl; and A representshydrogen or a non-interfering substituent (i.e. a substituent that doesnot adversely affect the ability of an oligomer to bind to its intendedtarget) on one or more carbon atoms in (b1′) and (b1″). Preferably, thering carbons in the piperazine ring are unsubstituted; however, the ringcarbons of the piperazine ring may include non-interfering substituents,such as methyl or fluorine. Preferably, at most one or two carbon atomsis so substituted.

In further embodiments, at least 10% of the linkages are of type (b1′)or (b1″); for example, 10%-60% and preferably 20% to 50%, of thelinkages may be of type (b1′) or (b1″).

In other embodiments, the oligomer contains no linkages of the type(b1′) above. Alternatively, the oligomer contains no linkages of type(b1) where each R is H, R³ is H or —CH₃, and R⁴ is H, —CH₃, or anelectron pair.

The morpholino subunits may also be linked by non-phosphorus-basedintersubunit linkages, as described further below, where at least onelinkage is modified with a pendant cationic group as described above.

Other oligonucleotide analog linkages which are uncharged in theirunmodified state but which could also bear a pendant amine substituentcould be used. For example, a 5′nitrogen atom on a morpholino ring couldbe employed in a sulfamide linkage or a urea linkage (where phosphorusis replaced with carbon or sulfur, respectively) and modified in amanner analogous to the 5′-nitrogen atom in structure (b3) above.

Oligomers having any number of cationic linkages are provided, includingfully cationic-linked oligomers. Preferably, however, the oligomers arepartially charged, having, for example, 10%-80%. In preferredembodiments, about 10% to 60%, and preferably 20% to 50% of the linkagesare cationic.

In one embodiment, the cationic linkages are interspersed along thebackbone. The partially charged oligomers preferably contain at leasttwo consecutive uncharged linkages; that is, the oligomer preferablydoes not have a strictly alternating pattern along its entire length.

Also considered are oligomers having blocks of cationic linkages andblocks of uncharged linkages; for example, a central block of unchargedlinkages may be flanked by blocks of cationic linkages, or vice versa.In one embodiment, the oligomer has approximately equal-length 5′, 3′and center regions, and the percentage of cationic linkages in thecenter region is greater than about 50%, preferably greater than about70%.

Oligomers for use in antisense applications generally range in lengthfrom about 10 to about 40 subunits, more preferably about 10 to 30subunits, and typically 15-25 bases, including those having 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 bases. In certain embodiments,an oligomer of the invention having 19-20 subunits, a useful length foran antisense compound, may ideally have two to ten, e.g., four to eight,cationic linkages, and the remainder uncharged linkages. An oligomerhaving 14-15 subunits may ideally have two to seven, e.g., 3 to 5,cationic linkages, and the remainder uncharged linkages.

Each morpholino ring structure supports a base pairing moiety, to form asequence of base pairing moieties which is typically designed tohybridize to a selected antisense target in a cell or in a subject beingtreated. The base pairing moiety may be a purine or pyrimidine found innative DNA or RNA (e.g., A, G, C, T or U) or an analog, such ashypoxanthine (the base component of the nucleoside inosine) or 5-methylcytosine.

Peptide Transporters

The antisense compounds of the invention may include an oligonucleotidemoiety conjugated to an arginine-rich peptide transport moiety effectiveto enhance transport of the compound into cells. The transport moiety ispreferably attached to a terminus of the oligomer, as shown, forexample, in FIGS. 1B and 1C. The peptide transport moiety preferablycomprises 6 to 16 subunits selected from X′ subunits, Y′ subunits, andZ′ subunits, wherein:

(a) each X′ subunit independently represents lysine, arginine or anarginine analog, said analog being a cationic α-amino acid comprising aside chain of the structure R¹N═C(NH₂)R², where R¹ is H or R; R² is R,—NH₂, —NHR, or —NR₂, where R is optionally substituted lower alkyl oroptionally substituted lower alkenyl; R¹ and R² may join together toform a ring; and the side chain is linked to said amino acid via R¹ orR²;

(b) each Y′ subunit independently represents a neutral amino acid—C(═O)—(CHR)_(n)—NH—, where n is 2 to 7 and each R is independently H ormethyl; and

(c) each Z′ subunit independently represents an α-amino acid having aneutral aralkyl side chain;

wherein the peptide comprises a sequence represented by at least one of(X′Y′X′)_(p), (X′Y′)_(m), and/or (X′Z′Z′)_(p), where p is 2 to 5 and mis 2 to 8. Certain embodiments include various combinations selectedindependently from (X′Y′X′)_(p), (X′Y′)_(m), and/or (X′Z′Z′)_(p),including, for example, peptides having the sequence(X′Y′X′)(X′Z′Z′)(X′Y′X′)(X′Z′Z′) (SEQ ID NO:637).

In selected embodiments, for each X′, the side chain moiety is guanidyl,as in the amino acid subunit arginine (Arg). In certain embodiments,each Y′ is independently —C(═O)—(CH₂)_(n)—CHR—NH—, where n is 2 to 7 andR is H. For example, when n is 5 and R is H, Y′ is a 6-aminohexanoicacid subunit, abbreviated herein as Ahx; when n is 2 and R is H, Y′ is aβ-alanine subunit, abbreviated herein as B. Certain embodiments relateto carrier peptides having a combination of different neutral aminoacids, including, for example, peptides comprising the sequence-RahxRRBRRAhxRRBRAhxB- (SEQ ID NO:578), which contains both β-alanineand 6-aminohexanoic acid.

Preferred peptides of this type include those comprising arginine dimersalternating with single Y′ subunits, where Y′ is preferably Ahx or B orboth. Examples include peptides having the formula (RY′R)_(p) and/or theformula (RRY′)_(p), where p is 1 to 2 to 5 and where Y′ is preferablyAhx. In one embodiment, Y′ is a 6-aminohexanoic acid subunit, R isarginine and p is 4. Certain embodiments include various linearcombinations of at least two of (RY′R)_(p) and (RRY′)_(p), including,for example, illustrative peptides having the sequence(RY′R)(RRY′)(RY′R)(RRY′) (SEQ ID NO:638), or (RRY′)(RY′R)(RRY′) (SEQ IDNO:639). Other combinations are contemplated. In a further illustrativeembodiment, each Z′ is phenylalanine, and m is 3 or 4.

The conjugated peptide is preferably linked to a terminus of theoligomer via a linker Ahx-B, where Ahx is a 6-aminohexanoic acid subunitand B is a β-alanine subunit, as shown, for example, in FIGS. 1B and 1C.

In selected embodiments, for each X′, the side chain moiety isindependently selected from the group consisting of guanidyl(HN═C(NH₂)NH—), amidinyl (HN═C(NH₂)CH—), 2-aminodihydropyrimidyl,2-aminotetrahydropyrimidyl, 2-aminopyridinyl, and 2-aminopyrimidonyl,and it is preferably selected from guanidyl and amidinyl. In oneembodiment, the side chain moiety is guanidyl, as in the amino acidsubunit arginine (Arg).

In certain embodiments, the Y′ subunits may be contiguous, in that no X′subunits intervene between Y′ subunits, or interspersed singly betweenX′ subunits. In certain embodiments, the linking subunit may be betweenY′ subunits. In one embodiment, the Y′ subunits are at a terminus of thetransporter; in other embodiments, they are flanked by X′ subunits. Infurther preferred embodiments, each Y′ is —C(═O)—(CH₂)_(n)—CHR—NH—,where n is 2 to 7 and R is H. For example, when n is 5 and R is H, Y′ isa 6-aminohexanoic acid subunit, abbreviated herein as Ahx. In selectedembodiments of this group, each X′ comprises a guanidyl side chainmoiety, as in an arginine subunit. Preferred peptides of this typeinclude those comprising arginine dimers alternating with single Y′subunits, where Y′ is preferably Ahx. Examples include peptides havingthe formula (RY′R)₄ or the formula (RRY′)₄, where Y′ is preferably Ahx.In the latter case, the nucleic acid analog is preferably linked to aterminal Y′ subunit, preferably at the C-terminus, as shown, forexample, in FIGS. 1B and 1C. The preferred linker is of the structureAhxB, where Ahx is a 6-aminohexanoic acid subunit and B is a β-alaninesubunit.

The transport moieties as described above have been shown to greatlyenhance cell entry of attached oligomers, relative to uptake of theoligomer in the absence of the attached transport moiety, and relativeto uptake by an attached transport moiety lacking the hydrophobicsubunits Y′. Such enhanced uptake is preferably evidenced by at least atwo-fold increase, and preferably a four-fold increase, in the uptake ofthe compound into mammalian cells relative to uptake of the agent by anattached transport moiety lacking the hydrophobic subunits Y′. Uptake ispreferably enhanced at least twenty fold, and more preferably fortyfold, relative to the unconjugated compound.

A further benefit of the transport moiety is its expected ability tostabilize a duplex between an antisense compound and its target nucleicacid sequence, presumably by virtue of electrostatic interaction betweenthe positively charged transport moiety and the negatively chargednucleic acid. The number of charged subunits in the transporter is lessthan 14, as noted above, and preferably between 8 and 11, since too higha number of charged subunits may lead to a reduction in sequencespecificity.

The use of arginine-rich peptide transporters (i.e., cell-penetratingpeptides) is particularly useful in practicing the present invention.Certain peptide transporters have been shown to be highly effective atdelivery of antisense compounds into primary cells including musclecells (Marshall, Oda et al. 2007; Jearawiriyapaisarn, Moulton et al.2008; Wu, Moulton et al. 2008). Furthermore, compared to other peptidetransporters such as Penetratin and the Tat peptide, the peptidetransporters described herein, when conjugated to an antisense PMO,demonstrate an enhanced ability to alter splicing of several genetranscripts (Marshall, Oda et al. 2007). Especially preferred are theP007, CP06062 and CP04057 transport peptides listed below in Table 3(SEQ ID NOS: 573, 578 and 577, respectively).

Exemplary peptide transporters, including linkers (B or AhxB) are givenbelow in Table B below. Preferred sequences are those designated CP06062(SEQ ID NO: 578), P007 (SEQ ID NO: 573) and CP04057 (SEQ ID NO: 577).

TABLE B Exemplary Peptide Transporters for Intracellular Delivery of PMOSEQ Sequence (N-terminal to ID Peptide C-terminal) NO: rTAT RRRQRRKKRC570 R₉F₂ RRRRRRRRRFFC 571 (RRAhx)₄B RRAhxRRAhxRRAhxRRAhxB 572(RAhxR)₄AhxB;  RAhxRRAhxRRAhxRRAhxRAhxB 573 (P007) (AhxRR)₄AhxBAhxRRAhxRRAhxRRAhxRRAhxB 574 (RAhx)₆B RAhxRAhxRAhxRAhxRAhxRAhxB 575(RAhx)₈B RAhxRAhxRAhxRAhxRAhxRAhxRAhx 576 B (RAhxR)₅AhxBRAhxRRAhxRRAhxRRAhxRRAhxRAhx 577 (CP05057) B (RAhxRRBR)₂AhxB;RAhxRRBRRAhxRRBRAhxB 578 (CP06062) MSP ASSLNIA 579Formulations

In certain embodiments, the present invention provides formulations orcompositions suitable for the therapeutic delivery of antisenseoligomers, as described herein. Hence, in certain embodiments, thepresent invention provides pharmaceutically acceptable compositions thatcomprise a therapeutically-effective amount of one or more of theoligomers described herein, formulated together with one or morepharmaceutically acceptable carriers (additives) and/or diluents. Whileit is possible for an oligomer of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical formulation (composition).

Methods for the delivery of nucleic acid molecules are described, forexample, in Akhtar et al., 1992, Trends Cell Bio., 2:139; and DeliveryStrategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar;Sullivan et al., PCT WO 94/02595. These and other protocols can beutilized for the delivery of virtually any nucleic acid molecule,including the isolated oligomers of the present invention.

As detailed below, the pharmaceutical compositions of the presentinvention may be specially formulated for administration in solid orliquid form, including those adapted for the following: (1) oraladministration, for example, drenches (aqueous or non-aqueous solutionsor suspensions), tablets, e.g., those targeted for buccal, sublingual,and systemic absorption, boluses, powders, granules, pastes forapplication to the tongue; (2) parenteral administration, for example,by subcutaneous, intramuscular, intravenous or epidural injection as,for example, a sterile solution or suspension, or sustained-releaseformulation; (3) topical application, for example, as a cream, ointment,or a controlled-release patch or spray applied to the skin; (4)intravaginally or intrarectally, for example, as a pessary, cream orfoam; (5) sublingually; (6) ocularly; (7) transdermally; or (8) nasally.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the patient.

Some examples of materials that can serve as pharmaceutically-acceptablecarriers include, without limitation: (1) sugars, such as lactose,glucose and sucrose; (2) starches, such as corn starch and potatostarch; (3) cellulose, and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate; (4) powderedtragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such ascocoa butter and suppository waxes; (9) oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; and (22) othernon-toxic compatible substances employed in pharmaceutical formulations.

Additional non-limiting examples of agents suitable for formulation withthe antisense oligomers of the instant invention include: PEG conjugatednucleic acids, phospholipid conjugated nucleic acids, nucleic acidscontaining lipophilic moieties, phosphorothioates, P-glycoproteininhibitors (such as Pluronic P85) which can enhance entry of drugs intovarious tissues; biodegradable polymers, such as poly(DL-lactide-coglycolide) microspheres for sustained release deliveryafter implantation (Emerich, D F et al., 1999, Cell Transplant, 8,47-58) Alkermes, Inc. Cambridge, Mass.; and loaded nanoparticles, suchas those made of polybutylcyanoacrylate, which can deliver drugs acrossthe blood brain barrier and can alter neuronal uptake mechanisms (ProgNeuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999).

The invention also features the use of the composition comprisingsurface-modified liposomes containing poly (ethylene glycol) lipids(PEG-modified, branched and unbranched or combinations thereof, orlong-circulating liposomes or stealth liposomes). Oligomers of theinvention can also comprise covalently attached PEG molecules of variousmolecular weights. These formulations offer a method for increasing theaccumulation of drugs in target tissues. This class of drug carriersresists opsonization and elimination by the mononuclear phagocyticsystem (MPS or RES), thereby enabling longer blood circulation times andenhanced tissue exposure for the encapsulated drug (Lasic et al. Chem.Rev. 1995, 95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43,1005-1011). Such liposomes have been shown to accumulate selectively intumors, presumably by extravasation and capture in the neovascularizedtarget tissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et al.,1995, Biochim. Biophys. Acta, 1238, 86-90). The long-circulatingliposomes enhance the pharmacokinetics and pharmacodynamics of DNA andRNA, particularly compared to conventional cationic liposomes which areknown to accumulate in tissues of the MPS (Liu et al., J. Biol. Chem.1995, 42, 24864-24870; Choi et al., International PCT Publication No. WO96/10391; Ansell et al., International PCT Publication No. WO 96/10390;Holland et al., International PCT Publication No. WO 96/10392).Long-circulating liposomes are also likely to protect drugs fromnuclease degradation to a greater extent compared to cationic liposomes,based on their ability to avoid accumulation in metabolically aggressiveMPS tissues such as the liver and spleen.

In a further embodiment, the present invention includes oligomercompositions prepared for delivery as described in U.S. Pat. Nos.6,692,911, 7,163,695 and 7,070,807. In this regard, in one embodiment,the present invention provides an oligomer of the present invention in acomposition comprising copolymers of lysine and histidine (HK) asdescribed in U.S. Pat. Nos. 7,163,695, 7,070,807, and 6,692,911 eitheralone or in combination with PEG (e.g., branched or unbranched PEG or amixture of both), in combination with PEG and a targeting moiety or anyof the foregoing in combination with a crosslinking agent. In certainembodiments, the present invention provides antisense oligomers incompositions comprising gluconic-acid-modified polyhistidine orgluconylated-polyhistidine/transferrin-polylysine. One skilled in theart will also recognize that amino acids with properties similar to Hisand Lys may be substituted within the composition.

Certain embodiments of the oligomers described herein may contain abasic functional group, such as amino or alkylamino, and are, thus,capable of forming pharmaceutically-acceptable salts withpharmaceutically-acceptable acids. The term “pharmaceutically-acceptablesalts” in this respect, refers to the relatively non-toxic, inorganicand organic acid addition salts of compounds of the present invention.These salts can be prepared in situ in the administration vehicle or thedosage form manufacturing process, or by separately reacting a purifiedcompound of the invention in its free base form with a suitable organicor inorganic acid, and isolating the salt thus formed during subsequentpurification. Representative salts include the hydrobromide,hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate,valerate, oleate, palmitate, stearate, laurate, benzoate, lactate,phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate,napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonatesalts and the like. (See, e.g., Berge et al. (1977) “PharmaceuticalSalts”, J. Pharm. Sci. 66:1-19).

The pharmaceutically acceptable salts of the subject oligomers includethe conventional nontoxic salts or quaternary ammonium salts of thecompounds, e.g., from non-toxic organic or inorganic acids. For example,such conventional nontoxic salts include those derived from inorganicacids such as hydrochloride, hydrobromic, sulfuric, sulfamic,phosphoric, nitric, and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicyclic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic, and the like.

In certain embodiments, the oligomers of the present invention maycontain one or more acidic functional groups and, thus, are capable offorming pharmaceutically-acceptable salts withpharmaceutically-acceptable bases. The term “pharmaceutically-acceptablesalts” in these instances refers to the relatively non-toxic, inorganicand organic base addition salts of compounds of the present invention.These salts can likewise be prepared in situ in the administrationvehicle or the dosage form manufacturing process, or by separatelyreacting the purified compound in its free acid form with a suitablebase, such as the hydroxide, carbonate or bicarbonate of apharmaceutically-acceptable metal cation, with ammonia, or with apharmaceutically-acceptable organic primary, secondary or tertiaryamine. Representative alkali or alkaline earth salts include thelithium, sodium, potassium, calcium, magnesium, and aluminum salts andthe like. Representative organic amines useful for the formation of baseaddition salts include ethylamine, diethylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine and the like. (See, e.g., Bergeet al., supra).

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral,nasal, topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient that canbe combined with a carrier material to produce a single dosage form willvary depending upon the host being treated, the particular mode ofadministration. The amount of active ingredient which can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the compound which produces a therapeutic effect.Generally, out of one hundred percent, this amount will range from about0.1 percent to about ninety-nine percent of active ingredient,preferably from about 5 percent to about 70 percent, most preferablyfrom about 10 percent to about 30 percent.

In certain embodiments, a formulation of the present invention comprisesan excipient selected from cyclodextrins, celluloses, liposomes, micelleforming agents, e.g., bile acids, and polymeric carriers, e.g.,polyesters and polyanhydrides; and an oligomer of the present invention.In certain embodiments, an aforementioned formulation renders orallybioavailable an oligomer of the present invention.

Methods of preparing these formulations or compositions include the stepof bringing into association an oligomer of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. An oligomer of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules, trouches and thelike), the active ingredient may be mixed with one or morepharmaceutically-acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds and surfactants, such as poloxamer and sodium laurylsulfate; (7) wetting agents, such as, for example, cetyl alcohol,glycerol monostearate, and non-ionic surfactants; (8) absorbents, suchas kaolin and bentonite clay; (9) lubricants, such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, zinc stearate, sodium stearate, stearic acid, and mixturesthereof; (10) coloring agents; and (11) controlled release agents suchas crospovidone or ethyl cellulose. In the case of capsules, tablets andpills, the pharmaceutical compositions may also comprise bufferingagents. Solid compositions of a similar type may also be employed asfillers in soft and hard-shelled gelatin capsules using such excipientsas lactose or milk sugars, as well as high molecular weight polyethyleneglycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (e.g., gelatin or hydroxypropylmethyl cellulose), lubricant,inert diluent, preservative, disintegrant (for example, sodium starchglycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be formulated for rapid release,e.g., freeze-dried. They may be sterilized by, for example, filtrationthrough a bacteria-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions which can be dissolvedin sterile water, or some other sterile injectable medium immediatelybefore use. These compositions may also optionally contain opacifyingagents and may be of a composition that they release the activeingredient(s) only, or preferentially, in a certain portion of thegastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations for rectal or vaginal administration may be presented as asuppository, which may be prepared by mixing one or more compounds ofthe invention with one or more suitable nonirritating excipients orcarriers comprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

Formulations or dosage forms for the topical or transdermaladministration of an oligomer as provided herein include powders,sprays, ointments, pastes, creams, lotions, gels, solutions, patches andinhalants. The active oligomers may be mixed under sterile conditionswith a pharmaceutically-acceptable carrier, and with any preservatives,buffers, or propellants which may be required. The ointments, pastes,creams and gels may contain, in addition to an active compound of thisinvention, excipients, such as animal and vegetable fats, oils, waxes,paraffins, starch, tragacanth, cellulose derivatives, polyethyleneglycols, silicones, bentonites, silicic acid, talc and zinc oxide, ormixtures thereof.

Powders and sprays can contain, in addition to an oligomer of thepresent invention, excipients such as lactose, talc, silicic acid,aluminum hydroxide, calcium silicates and polyamide powder, or mixturesof these substances. Sprays can additionally contain customarypropellants, such as chlorofluorohydrocarbons and volatile unsubstitutedhydrocarbons, such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of an oligomer of the present invention to the body. Suchdosage forms can be made by dissolving or dispersing the oligomer in theproper medium. Absorption enhancers can also be used to increase theflux of the agent across the skin. The rate of such flux can becontrolled by either providing a rate controlling membrane or dispersingthe agent in a polymer matrix or gel, among other methods known in theart.

Pharmaceutical compositions suitable for parenteral administration maycomprise one or more oligomers of the invention in combination with oneor more pharmaceutically-acceptable sterile isotonic aqueous ornonaqueous solutions, dispersions, suspensions or emulsions, or sterilepowders which may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain sugars, alcohols,antioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents. Examples of suitable aqueous andnonaqueous carriers which may be employed in the pharmaceuticalcompositions of the invention include water, ethanol, polyols (such asglycerol, propylene glycol, polyethylene glycol, and the like), andsuitable mixtures thereof, vegetable oils, such as olive oil, andinjectable organic esters, such as ethyl oleate. Proper fluidity can bemaintained, for example, by the use of coating materials, such aslecithin, by the maintenance of the required particle size in the caseof dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms upon the subject oligomers may be ensuredby the inclusion of various antibacterial and antifungal agents, forexample, paraben, chlorobutanol, phenol sorbic acid, and the like. Itmay also be desirable to include isotonic agents, such as sugars, sodiumchloride, and the like into the compositions. In addition, prolongedabsorption of the injectable pharmaceutical form may be brought about bythe inclusion of agents which delay absorption such as aluminummonostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility, amongother methods known in the art. The rate of absorption of the drug thendepends upon its rate of dissolution which, in turn, may depend uponcrystal size and crystalline form. Alternatively, delayed absorption ofa parenterally-administered drug form is accomplished by dissolving orsuspending the drug in an oil vehicle.

Injectable depot forms may be made by forming microencapsule matrices ofthe subject oligomers in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of oligomer topolymer, and the nature of the particular polymer employed, the rate ofoligomer release can be controlled. Examples of other biodegradablepolymers include poly(orthoesters) and poly(anhydrides). Depotinjectable formulations may also prepared by entrapping the drug inliposomes or microemulsions that are compatible with body tissues.

When the oligomers of the present invention are administered aspharmaceuticals, to humans and animals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99% (morepreferably, 10 to 30%) of active ingredient in combination with apharmaceutically acceptable carrier.

As noted above, the formulations or preparations of the presentinvention may be given orally, parenterally, topically, or rectally.They are typically given in forms suitable for each administrationroute. For example, they are administered in tablets or capsule form, byinjection, inhalation, eye lotion, ointment, suppository, etc.administration by injection, infusion or inhalation; topical by lotionor ointment; and rectal by suppositories.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

Regardless of the route of administration selected, the oligomers of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, may beformulated into pharmaceutically-acceptable dosage forms by conventionalmethods known to those of skill in the art. Actual dosage levels of theactive ingredients in the pharmaceutical compositions of this inventionmay be varied so as to obtain an amount of the active ingredient whichis effective to achieve the desired therapeutic response for aparticular patient, composition, and mode of administration, withoutbeing unacceptably toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular oligomer of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion ormetabolism of the particular oligomer being employed, the rate andextent of absorption, the duration of the treatment, other drugs,compounds and/or materials used in combination with the particularoligomer employed, the age, sex, weight, condition, general health andprior medical history of the patient being treated, and like factorswell known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved. In general, a suitabledaily dose of a compound of the invention will be that amount of thecompound which is the lowest dose effective to produce a therapeuticeffect. Such an effective dose will generally depend upon the factorsdescribed above. Generally, oral, intravenous, intracerebroventricularand subcutaneous doses of the compounds of this invention for a patient,when used for the indicated effects, will range from about 0.0001 toabout 100 mg per kilogram of body weight per day.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms. In certain situations, dosing is oneadministration per day. In certain embodiments, dosing is one or moreadministration per every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14days, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks, or every 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, as needed, to maintain thedesired expression of a functional dystrophin protein.

Nucleic acid molecules can be administered to cells by a variety ofmethods known to those familiar to the art, including, but notrestricted to, encapsulation in liposomes, by iontophoresis, or byincorporation into other vehicles, such as hydrogels, cyclodextrins,biodegradable nanocapsules, and bioadhesive microspheres, as describedherein and known in the art. In certain embodiments, microemulsificationtechnology may be utilized to improve bioavailability of lipophilic(water insoluble) pharmaceutical agents. Examples include Trimetrine(Dordunoo, S. K., et al., Drug Development and Industrial Pharmacy,17(12), 1685-1713, 1991 and REV 5901 (Sheen, P. C., et al., J Pharm Sci80(7), 712-714, 1991). Among other benefits, microemulsificationprovides enhanced bioavailability by preferentially directing absorptionto the lymphatic system instead of the circulatory system, which therebybypasses the liver, and prevents destruction of the compounds in thehepatobiliary circulation.

In one aspect of invention, the formulations contain micelles formedfrom an oligomer as provided herein and at least one amphiphiliccarrier, in which the micelles have an average diameter of less thanabout 100 nm. More preferred embodiments provide micelles having anaverage diameter less than about 50 nm, and even more preferredembodiments provide micelles having an average diameter less than about30 nm, or even less than about 20 nm.

While all suitable amphiphilic carriers are contemplated, the presentlypreferred carriers are generally those that haveGenerally-Recognized-as-Safe (GRAS) status, and that can both solubilizethe compound of the present invention and microemulsify it at a laterstage when the solution comes into a contact with a complex water phase(such as one found in human gastro-intestinal tract). Usually,amphiphilic ingredients that satisfy these requirements have HLB(hydrophilic to lipophilic balance) values of 2-20, and their structurescontain straight chain aliphatic radicals in the range of C-6 to C-20.Examples are polyethylene-glycolized fatty glycerides and polyethyleneglycols.

Examples of amphiphilic carriers include saturated and monounsaturatedpolyethyleneglycolyzed fatty acid glycerides, such as those obtainedfrom fully or partially hydrogenated various vegetable oils. Such oilsmay advantageously consist of tri-, di-, and mono-fatty acid glyceridesand di- and mono-polyethyleneglycol esters of the corresponding fattyacids, with a particularly preferred fatty acid composition includingcapric acid 4-10, capric acid 3-9, lauric acid 40-50, myristic acid14-24, palmitic acid 4-14 and stearic acid 5-15%. Another useful classof amphiphilic carriers includes partially esterified sorbitan and/orsorbitol, with saturated or mono-unsaturated fatty acids (SPAN-series)or corresponding ethoxylated analogs (TWEEN-series).

Commercially available amphiphilic carriers may be particularly useful,including Gelucire-series, Labrafil, Labrasol, or Lauroglycol (allmanufactured and distributed by Gattefosse Corporation, Saint Priest,France), PEG-mono-oleate, PEG-di-oleate, PEG-mono-laurate anddi-laurate, Lecithin, Polysorbate 80, etc (produced and distributed by anumber of companies in USA and worldwide).

In certain embodiments, the delivery may occur by use of liposomes,nanocapsules, microparticles, microspheres, lipid particles, vesicles,and the like, for the introduction of the compositions of the presentinvention into suitable host cells. In particular, the compositions ofthe present invention may be formulated for delivery either encapsulatedin a lipid particle, a liposome, a vesicle, a nanosphere, a nanoparticleor the like. The formulation and use of such delivery vehicles can becarried out using known and conventional techniques.

Hydrophilic polymers suitable for use in the present invention are thosewhich are readily water-soluble, can be covalently attached to avesicle-forming lipid, and which are tolerated in vivo without toxiceffects (i.e., are biocompatible). Suitable polymers includepolyethylene glycol (PEG), polylactic (also termed polylactide),polyglycolic acid (also termed polyglycolide), a polylactic-polyglycolicacid copolymer, and polyvinyl alcohol. In certain embodiments, polymershave a molecular weight of from about 100 or 120 daltons up to about5,000 or 10,000 daltons, or from about 300 daltons to about 5,000daltons. In other embodiments, the polymer is polyethyleneglycol havinga molecular weight of from about 100 to about 5,000 daltons, or having amolecular weight of from about 300 to about 5,000 daltons. In certainembodiments, the polymer is polyethyleneglycol of 750 daltons(PEG(750)). Polymers may also be defined by the number of monomerstherein; a preferred embodiment of the present invention utilizespolymers of at least about three monomers, such PEG polymers consistingof three monomers (approximately 150 daltons).

Other hydrophilic polymers which may be suitable for use in the presentinvention include polyvinylpyrrolidone, polymethoxazoline,polyethyloxazoline, polyhydroxypropyl methacrylamide,polymethacrylamide, polydimethylacrylamide, and derivatized cellulosessuch as hydroxymethylcellulose or hydroxyethylcellulose.

In certain embodiments, a formulation of the present invention comprisesa biocompatible polymer selected from the group consisting ofpolyamides, polycarbonates, polyalkylenes, polymers of acrylic andmethacrylic esters, polyvinyl polymers, polyglycolides, polysiloxanes,polyurethanes and copolymers thereof, celluloses, polypropylene,polyethylenes, polystyrene, polymers of lactic acid and glycolic acid,polyanhydrides, poly(ortho)esters, poly(butic acid), poly(valeric acid),poly(lactide-co-caprolactone), polysaccharides, proteins, polyhyaluronicacids, polycyanoacrylates, and blends, mixtures, or copolymers thereof.

Cyclodextrins are cyclic oligosaccharides, consisting of 6, 7 or 8glucose units, designated by the Greek letter α, β. or γ, respectively.The glucose units are linked by α-1,4-glucosidic bonds. As a consequenceof the chair conformation of the sugar units, all secondary hydroxylgroups (at C-2, C-3) are located on one side of the ring, while all theprimary hydroxyl groups at C-6 are situated on the other side. As aresult, the external faces are hydrophilic, making the cyclodextrinswater-soluble. In contrast, the cavities of the cyclodextrins arehydrophobic, since they are lined by the hydrogen of atoms C-3 and C-5,and by ether-like oxygens. These matrices allow complexation with avariety of relatively hydrophobic compounds, including, for instance,steroid compounds such as 17α-estradiol (see, e.g., van Uden et al.Plant Cell Tiss. Org. Cult. 38:1-3-113 (1994)). The complexation takesplace by Van der Waals interactions and by hydrogen bond formation. Fora general review of the chemistry of cyclodextrins, see, Wenz, Agnew.Chem. Int. Ed. Engl., 33:803-822 (1994).

The physico-chemical properties of the cyclodextrin derivatives dependstrongly on the kind and the degree of substitution. For example, theirsolubility in water ranges from insoluble (e.g.,triacetyl-beta-cyclodextrin) to 147% soluble (w/v)(G-2-beta-cyclodextrin). In addition, they are soluble in many organicsolvents. The properties of the cyclodextrins enable the control oversolubility of various formulation components by increasing or decreasingtheir solubility.

Numerous cyclodextrins and methods for their preparation have beendescribed. For example, Parmeter (I), et al. (U.S. Pat. No. 3,453,259)and Gramera, et al. (U.S. Pat. No. 3,459,731) described electroneutralcyclodextrins. Other derivatives include cyclodextrins with cationicproperties [Parmeter (II), U.S. Pat. No. 3,453,257], insolublecrosslinked cyclodextrins (Solms, U.S. Pat. No. 3,420,788), andcyclodextrins with anionic properties [Parmeter (III), U.S. Pat. No.3,426,011]. Among the cyclodextrin derivatives with anionic properties,carboxylic acids, phosphorous acids, phosphinous acids, phosphonicacids, phosphoric acids, thiophosphonic acids, thiosulphinic acids, andsulfonic acids have been appended to the parent cyclodextrin [see,Parmeter (III), supra]. Furthermore, sulfoalkyl ether cyclodextrinderivatives have been described by Stella, et al. (U.S. Pat. No.5,134,127).

Liposomes consist of at least one lipid bilayer membrane enclosing anaqueous internal compartment. Liposomes may be characterized by membranetype and by size. Small unilamellar vesicles (SUVs) have a singlemembrane and typically range between 0.02 and 0.05 μm in diameter; largeunilamellar vesicles (LUVS) are typically larger than 0.05 μm.Oligolamellar large vesicles and multilamellar vesicles have multiple,usually concentric, membrane layers and are typically larger than 0.1μm. Liposomes with several nonconcentric membranes, i.e., severalsmaller vesicles contained within a larger vesicle, are termedmultivesicular vesicles.

One aspect of the present invention relates to formulations comprisingliposomes containing an oligomer of the present invention, where theliposome membrane is formulated to provide a liposome with increasedcarrying capacity. Alternatively or in addition, the compound of thepresent invention may be contained within, or adsorbed onto, theliposome bilayer of the liposome. An oligomer of the present inventionmay be aggregated with a lipid surfactant and carried within theliposome's internal space; in these cases, the liposome membrane isformulated to resist the disruptive effects of the activeagent-surfactant aggregate.

According to one embodiment of the present invention, the lipid bilayerof a liposome contains lipids derivatized with polyethylene glycol(PEG), such that the PEG chains extend from the inner surface of thelipid bilayer into the interior space encapsulated by the liposome, andextend from the exterior of the lipid bilayer into the surroundingenvironment.

Active agents contained within liposomes of the present invention are insolubilized form. Aggregates of surfactant and active agent (such asemulsions or micelles containing the active agent of interest) may beentrapped within the interior space of liposomes according to thepresent invention. A surfactant acts to disperse and solubilize theactive agent, and may be selected from any suitable aliphatic,cycloaliphatic or aromatic surfactant, including but not limited tobiocompatible lysophosphatidylcholines (LPGs) of varying chain lengths(for example, from about C14 to about C20). Polymer-derivatized lipidssuch as PEG-lipids may also be utilized for micelle formation as theywill act to inhibit micelle/membrane fusion, and as the addition of apolymer to surfactant molecules decreases the CMC of the surfactant andaids in micelle formation. Preferred are surfactants with CMOs in themicromolar range; higher CMC surfactants may be utilized to preparemicelles entrapped within liposomes of the present invention.

Liposomes according to the present invention may be prepared by any of avariety of techniques that are known in the art. See, e.g., U.S. Pat.No. 4,235,871; Published PCT applications WO 96/14057; New RRC,Liposomes: A practical approach, IRL Press, Oxford (1990), pages 33-104;Lasic DD, Liposomes from physics to applications, Elsevier SciencePublishers BV, Amsterdam, 1993. For example, liposomes of the presentinvention may be prepared by diffusing a lipid derivatized with ahydrophilic polymer into preformed liposomes, such as by exposingpreformed liposomes to micelles composed of lipid-grafted polymers, atlipid concentrations corresponding to the final mole percent ofderivatized lipid which is desired in the liposome. Liposomes containinga hydrophilic polymer can also be formed by homogenization, lipid-fieldhydration, or extrusion techniques, as are known in the art.

In another exemplary formulation procedure, the active agent is firstdispersed by sonication in a lysophosphatidylcholine or other low CMCsurfactant (including polymer grafted lipids) that readily solubilizeshydrophobic molecules. The resulting micellar suspension of active agentis then used to rehydrate a dried lipid sample that contains a suitablemole percent of polymer-grafted lipid, or cholesterol. The lipid andactive agent suspension is then formed into liposomes using extrusiontechniques as are known in the art, and the resulting liposomesseparated from the unencapsulated solution by standard columnseparation.

In one aspect of the present invention, the liposomes are prepared tohave substantially homogeneous sizes in a selected size range. Oneeffective sizing method involves extruding an aqueous suspension of theliposomes through a series of polycarbonate membranes having a selecteduniform pore size; the pore size of the membrane will correspond roughlywith the largest sizes of liposomes produced by extrusion through thatmembrane. See e.g., U.S. Pat. No. 4,737,323 (Apr. 12, 1988). In certainembodiments, reagents such as DharmaFECT® and Lipofectamine® may beutilized to introduce polynucleotides or proteins into cells.

The release characteristics of a formulation of the present inventiondepend on the encapsulating material, the concentration of encapsulateddrug, and the presence of release modifiers. For example, release can bemanipulated to be pH dependent, for example, using a pH sensitivecoating that releases only at a low pH, as in the stomach, or a higherpH, as in the intestine. An enteric coating can be used to preventrelease from occurring until after passage through the stomach. Multiplecoatings or mixtures of cyanamide encapsulated in different materialscan be used to obtain an initial release in the stomach, followed bylater release in the intestine. Release can also be manipulated byinclusion of salts or pore forming agents, which can increase wateruptake or release of drug by diffusion from the capsule. Excipientswhich modify the solubility of the drug can also be used to control therelease rate. Agents which enhance degradation of the matrix or releasefrom the matrix can also be incorporated. They can be added to the drug,added as a separate phase (i.e., as particulates), or can beco-dissolved in the polymer phase depending on the compound. In mostcases the amount should be between 0.1 and thirty percent (w/w polymer).Types of degradation enhancers include inorganic salts such as ammoniumsulfate and ammonium chloride, organic acids such as citric acid,benzoic acid, and ascorbic acid, inorganic bases such as sodiumcarbonate, potassium carbonate, calcium carbonate, zinc carbonate, andzinc hydroxide, and organic bases such as protamine sulfate, spermine,choline, ethanolamine, diethanolamine, and triethanolamine andsurfactants such as Tween® and Pluronic®. Pore forming agents which addmicrostructure to the matrices (i.e., water soluble compounds such asinorganic salts and sugars) are added as particulates. The range istypically between one and thirty percent (w/w polymer).

Uptake can also be manipulated by altering residence time of theparticles in the gut. This can be achieved, for example, by coating theparticle with, or selecting as the encapsulating material, a mucosaladhesive polymer. Examples include most polymers with free carboxylgroups, such as chitosan, celluloses, and especially polyacrylates (asused herein, polyacrylates refers to polymers including acrylate groupsand modified acrylate groups such as cyanoacrylates and methacrylates).

An oligomer may be formulated to be contained within, or, adapted torelease by a surgical or medical device or implant. In certain aspects,an implant may be coated or otherwise treated with an oligomer. Forexample, hydrogels, or other polymers, such as biocompatible and/orbiodegradable polymers, may be used to coat an implant with thecompositions of the present invention (i.e., the composition may beadapted for use with a medical device by using a hydrogel or otherpolymer). Polymers and copolymers for coating medical devices with anagent are well-known in the art. Examples of implants include, but arenot limited to, stents, drug-eluting stents, sutures, prosthesis,vascular catheters, dialysis catheters, vascular grafts, prostheticheart valves, cardiac pacemakers, implantable cardioverterdefibrillators, IV needles, devices for bone setting and formation, suchas pins, screws, plates, and other devices, and artificial tissuematrices for wound healing.

In addition to the methods provided herein, the oligomers for useaccording to the invention may be formulated for administration in anyconvenient way for use in human or veterinary medicine, by analogy withother pharmaceuticals. The antisense oligomers and their correspondingformulations may be administered alone or in combination with othertherapeutic strategies in the treatment of muscular dystrophy, such asmyoblast transplantation, stem cell therapies, administration ofaminoglycoside antibiotics, proteasome inhibitors, and up-regulationtherapies (e.g., upregulation of utrophin, an autosomal paralogue ofdystrophin).

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims. The following examples are provided byway of illustration only and not by way of limitation. Those of skill inthe art will readily recognize a variety of noncritical parameters thatcould be changed or modified to yield essentially similar results.

REFERENCES

-   Aartsma-Rus, A., A. A. Janson, et al. (2004). “Antisense-induced    multiexon skipping for Duchenne muscular dystrophy makes more    sense.” Am J Hum Genet 74(1): 83-92.-   Dunckley, M. G., I. C. Eperon, et al. (1997). “Modulation of    splicing in the DMD gene by antisense oligoribonucleotides.”    Nucleosides & Nucleotides 16(7-9): 1665-1668.-   Dunckley, M. G., M. Manoharan, et al. (1998). “Modification of    splicing in the dystrophin gene in cultured Mdx muscle cells by    antisense oligoribonucleotides.” Hum Mol Genet 7(7): 1083-90.-   Errington, S. J., C. J. Mann, et al. (2003). “Target selection for    antisense oligonucleotide induced exon skipping in the dystrophin    gene.” J Gene Med 5(6): 518-27.-   Jearawiriyapaisarn, N., H. M. Moulton, et al. (2008). “Sustained    Dystrophin Expression Induced by Peptide-conjugated Morpholino    Oligomers in the Muscles of mdx Mice.” Mol Ther.-   Lu, Q. L., C. J. Mann, et al. (2003). “Functional amounts of    dystrophin produced by skipping the mutated exon in the mdx    dystrophic mouse.” Nat Med 9(8): 1009-14.-   Mann, C. J., K. Honeyman, et al. (2002). “Improved antisense    oligonucleotide induced exon skipping in the mdx mouse model of    muscular dystrophy.” J Gene Med 4(6): 644-54.-   Marshall, N. B., S. K. Oda, et al. (2007). “Arginine-rich    cell-penetrating peptides facilitate delivery of antisense oligomers    into murine leukocytes and alter pre-mRNA splicing.” Journal of    Immunological Methods 325(1-2): 114-126.-   Matsuo, M., T. Masumura, et al. (1991). “Exon skipping during    splicing of dystrophin mRNA precursor due to an intraexon deletion    in the dystrophin gene of Duchenne muscular dystrophy kobe.” J Clin    Invest 87(6): 2127-31.-   Monaco, A. P., C. J. Bertelson, et al. (1988). “An explanation for    the phenotypic differences between patients bearing partial    deletions of the DMD locus.” Genomics 2(1): 90-5.-   Pramono, Z. A., Y. Takeshima, et al. (1996). “Induction of exon    skipping of the dystrophin transcript in lymphoblastoid cells by    transfecting an antisense oligodeoxynucleotide complementary to an    exon recognition sequence.” Biochem Biophys Res Commun 226(2):    445-9.-   Sazani, P., R. Kole, et al. (2007). Splice switching oligomers for    the TNF superfamily receptors and their use in treatment of disease.    PCT WO2007058894, University of North Carolina-   Sierakowska, H., M. J. Sambade, et al. (1996). “Repair of    thalassemic human beta-globin mRNA in mammalian cells by antisense    oligonucleotides.” Proc Natl Acad Sci USA 93(23): 12840-4.-   Summerton, J. and D. Weller (1997). “Morpholino antisense oligomers:    design, preparation, and properties.” Antisense Nucleic Acid Drug    Dev 7(3): 187-95.-   Takeshima, Y., H. Nishio, et al. (1995). “Modulation of in vitro    splicing of the upstream intron by modifying an intra-exon sequence    which is deleted from the dystrophin gene in dystrophin Kobe.” J    Clin Invest 95(2): 515-20.-   van Deutekom, J. C., M. Bremmer-Bout, et al. (2001).    “Antisense-induced exon skipping restores dystrophin expression in    DMD patient derived muscle cells.” Hum Mol Genet 10(15): 1547-54.-   van Deutekom, J. C., A. A. Janson, et al. (2007). “Local dystrophin    restoration with antisense oligonucleotide PRO051.” N Engl J Med    357(26): 2677-86.-   Wilton, S. D., A. M. Fall, et al. (2007). “Antisense    oligonucleotide-induced exon skipping across the human dystrophin    gene transcript.” Mol Ther 15(7): 1288-96.-   Wilton, S. D., F. Lloyd, et al. (1999). “Specific removal of the    nonsense mutation from the mdx dystrophin mRNA using antisense    oligonucleotides.” Neuromuscul Disord 9(5): 330-8.-   Wu, B., H. M. Moulton, et al. (2008). “Effective rescue of    dystrophin improves cardiac function in dystrophin-deficient mice by    a modified morpholino oligomer.” Proc Natl Acad Sci USA 105(39):    14814-9.-   Yin, H., H. M. Moulton, et al. (2008). “Cell-penetrating    peptide-conjugated antisense oligonucleotides restore systemic    muscle and cardiac dystrophin expression and function.” Hum Mol    Genet 17(24): 3909-18.

EXAMPLES Materials and Methods Cells and Tissue Culture TreatmentConditions

Human Rhabdomyosarcoma cells (ATCC, CCL-136; RD cells) preserved in a 5%DMSO solution (Sigma) at a low passage number were thawed in a 37° C.water bath until the ice sliver was no longer visible. Cells were seededinto tissue culture-treated T75 flasks (Nunc) at 1.5×10⁶ cells/flask in24 mL of warmed DMEM with L-Glutamine (HyClone), 10% fetal bovine serum,and 1% Penicillin-Streptomycin antibiotic solution (CelGro); after 24hours, media was aspirated, cells were washed once in warmed PBS, andfresh media was added. Cells were grown to 80% confluence in a 37° C.incubator at 5.0% CO2.

Media was aspirated from T75 flasks; cells were washed once in warmedPBS and aspirated. 3 mL of Trypsin/EDTA, warmed in a 37° C. water bath,was added to each T75. Cells were incubated at 37° C. 5 2-5 minutesuntil, with gentle agitation, they released from the flask. Cellsuspension was transferred to a 15.0 mL conical tube; flasks were rinsedwith 1.0 mL of Trypsin/EDTA solution to gather remaining cells. Cellswere counted with a Vi-Cell XR cell counter (Beckman Coulter). Cellswere seeded into tissue culture-treated 12-well plates (Falcon) at2.0×10⁵ viable cells per well in 1.0 mL media. Cells were incubatedovernight in a 37° C. incubator at 5.0% CO₂.

Twelve-well seeded plates were examined for even cellular distributionand plate adherence. Lyophilized peptide conjugated phosphorodiamidatemorpholino oligomers (PPMOs) were re-suspended at 2.0 mM innuclease-free water (Ambion), and kept on ice during cell treatment; toverify molarity, PPMOs were measured using a NanoDrop 2000spectrophotometer (Thermo Scientific). Immediately prior to PPMOtreatment, media was aspirated, and cells were rinsed in warmed PBS.PPMOs were diluted in warmed media to the desired molarity; cells weretreated in a total of 1.0 mL PPMO per well. PPMOs were tested intriplicate. For no-treatment controls, fresh, warmed media was added in1.0 mL total volume. Cells were incubated for 48 hours in a 37° C.incubator at 5.0% CO2.

RNA Extraction

Media was aspirated, and cells were rinsed in warmed PBS. RNA wasextracted with the QuickGene-Mini80 system, QuickGene RNA cultured cellHC kit S, and MagNAlyser with ceramic bead homogenization using themanufacturers' recommended protocols. Briefly, cells were lysed intreatment plates with 350 uL LRP (10 uL β-Mercaptoethanol added per 100uL LRP) lysis buffer; homogenate was gently triturated to ensure fulllysis, and transferred to MagNAlyser tubes. Tubes were spun at 2800 rpmfor 30 seconds in the MagNAlyser to ensure full homogenization, and icedbriefly. 50 uL SRP solubilization buffer was added and homogenate wasvortexed for 15 seconds. 170 uL>99% ethanol was added to each tube, andhomogenate was vortexed for 60 seconds. Homogenate was flash-spun andtransferred to Mini80 RNA cartridges, samples were pressurized andflow-through was discarded. Cartridges were washed in 750 uL WRP washbuffer and pressurized. 40 uL of DNase solution (1.25 uL Qiagen DNasel,35 uL RDD Buffer, 3.75 uL nuclease-free water) was added directly to thecartridge membrane; cartridges were incubated four minutes at roomtemperature. Cartridges were washed twice with 750 uL WRP, pressurizingafter each wash. Cartridges were placed over nuclease-free tubes. 50 uLCRP elution buffer was added to each membrane; membranes were incubatedfor five minutes at room-temperature. Cartridges were pressurized andeluate was collected. RNA was stored at −80° C. pending quantification.RNA was quantified using the NanoDrop™ 2000 spectrophotometer.

Nested RT-PCR

Primer-specific, exon-specific, optimized nested RT-PCR amplificationwas performed using the primer pair sets for each dystrophin exon asshown below in Table 1.

TABLE 1 Primer pair sets used to PCR amplifyhuman dystrophin mRNA to detect exon-skipping. SEQ ID Name F/R I/OSequence (5′-3′) Exon Purpose NO: PS170 F O CCAGAGCTTTACCTGAGAAACAAG 48Detection 640 PS172 F I CCAGCCACTCAGCCAGTGAAG 49 of Exon 50 641 PS174 RI CGATCCGTAATGATTGTTCTAGCC 52 and 51 642 PS176 R OCATTTCATTCAACTGTTGCCTCCG 53 Skipping in 643 Human Dystrophin PS186 F OCAATGCTCCTGACCTCTGTGC 42 Detection 644 PS187 F I GTCTACAACAAAGCTCAGGTCG43 of Exon 44 645 PS189 F I GCAATGTTATCTGCTTCCTCCAACC 46 and 45 646PS190 R O GCTCTTTTCCAGGTTCAAGTGG 46 Skipping in 647 Human DystrophinPS192 F O CTTGGACAGAACTTACCGACTGG 51 Detection 648 PS193 F IGCAGGATTTGGAACAGAGGCG 52 of Exon 53 649 PS195 R ICATCTACATTTGTCTGCCACTGG 54 Skipping in 650 PS197 R OGTTTCTTCCAAAGCAGCCTCTCG 55 Human 651 Dystrophin

The indicated primer pairs are shown as either forward or reverse (F/R)and either outer or inner primer pairs (I/O) corresponding to primary orsecondary amplifications, respectively. The location of the primertarget is indicated in the Exon column and the Purpose indicates theexon-skipping events can be detected. For example, PS170 and PS176primers amplify a region from exon 48 to 53 in the primaryamplification. Primers PS172 and PS174 then amplify a region from exon49 to 52 in the secondary amplification. This nested PCR reaction willdetect exon skipping of both exons 50 and/or exon 51. The specificnested RT-PCR reaction conditions are provided below.

RNA extracted from treated cells (described above) was diluted to 20ng/ul for all samples.

TABLE 2 Reaction setup for RT-PCR and primary amplification (50 μlreaction): 2× Reaction mix 25 μl PS XXX Forward Primer (30 μM) 0.5 μl (see Table 1) PS XXX Reverse Primer (30 μM) 0.5 μl  (see Table 1)Superscript III Platinum Taq mix  2 μl Template RNA (20 ng/μl) 10 μlNuclease-Free Water (50 μl total 12 μl volume)

TABLE 3 RT-PCR and primary amplification program: Temperature TimeReverse 55° C. 30 minutes Transcription RT Inactivation 94° C. 2 minutesDenaturing 94° C. 1 minute 8 Cycles Annealing 59° C. 1 minute Extension68° C. 1 minute  4° C. ∞

TABLE 4 Reaction setup for nested secondary amplification (50 μlreaction): 10× PCR Buffer   5 μl dNTP solution (10 mM) 0.5 μl 50 mM MgCl1.5 μl PS XXX Forward Primer (30 μM) 0.33 μl  (see Table 1) PS XXXReverse Primer (30 μM) 0.33 μl  (see Table 1) Platinum Taq DNApolymerase 0.2 μl 0.1 mM Cy5-dCTP   1 μl RT-PCR product (from Step 1)  1 μl Nuclease-Free Water (50 μl total 40.15 μl  volume)

TABLE 5 Nested secondary amplification program: Temperature Time Primary94° C. 3 minutes Denature Denaturing 94° C. 45 seconds 28-30 Annealing59° C. 30 seconds Cycles Extension 68° C. 1 minute  4° C. ∞

Gel Electrophoresis Analysis

Ten microliters of 5× Ficoll loading dye was added to each 50 microliternested RT-PCR reaction. Fifteen microliters of PCR/dye mixture was runon a 10% TBE gel at 300 volts for 30 minutes. After electrophoresis, thegel was washed in diH2O for at least one hour, changing the water every30 minutes. The gel was then scanned on a Typhoon Trio Variable ModeImager (GE Healthcare). For exon 44 skipping, the nested RT-PCR productfrom full-length dystrophin transcript is 571 bp, and 423 bp from Exon44-skipped mRNA (exon 44 is 148 bp). For exon 45, the nested RT-PCRproduct from full-length dystrophin transcript is 571 bp, and 395 bpfrom Exon 45-skipped mRNA (exon 45 is 176 bp). For exon 53, the PCRproduct from full-length dystrophin transcript is 365 bp, and 153 bpfrom exon 53-skipped mRNA (exon 53 is 212 bp).

The gel images were subjected to quantitative analysis by measuring theband intensities of the full-length PCR product compared to theexon-skipped product. In some cases, the percent skipping at a fixedPPMO concentration (e.g., 3 micromolar) was used to determine therelative activity of a series of PPMO to induce exon skipping of a givenexon. In other situations, a PPMO dose-range was used to treat cells(e.g., 0.1, 0.3, 1.0, 3.0 and 10 micromolar) and an EC₅₀ was calculatedbased on the percent skipping induced at each concentration.

Example 1 Exon 51 Scan

A series of overlapping antisense PPMOs that target human dystrophinexon 51 were designed, synthesized and used to treat either humanrhabdomyosarcoma cells (RD cells) or primary human skeletal musclecells. This strategy is termed an “exon scan” and was used similarly forseveral other dystrophin exons as described below. All the PPMOs weresynthesized as peptide-conjugated PMO (PPMO) using the CP06062 peptide(SEQ ID NO: 578) and a 3′ terminal PMO linkage. For exon 51, a series of26 PPMOs, each 26 bases in length, were made (SEQ ID NOS: 309-311, 314,316, 317, 319, 321, 323, 324, 326, 327, 329-331, 333, 335, 336, 338-345)as shown in FIG. 2A. The PPMOs were evaluated for exon skipping efficacyby treating RD cells at various concentrations as described above in theMaterials and Methods. Three PPMOs (SEQ ID NOS: 324, 326 and 327) wereidentified as effective in inducing exon-skipping and selected foradditional evaluation. Dose-ranging experiments in RD cells and primaryhuman skeletal muscle cells were used to confirm the relative efficacyof these three PPMO sequences. SEQ ID NO: 327 was shown to be mosteffective at inducing exon 51 skipping as shown in FIGS. 2B and 2C.

A comparison of the relative effectiveness of SEQ ID NO: 327 to otherexon 51-targeted antisense sequences was performed in RD cells andprimary human skeletal muscle cells, as described above. All theevaluated sequences were made as peptide-conjugated PMOs using theCP06062 peptide (SEQ ID NO: 578). This allowed direct comparison of therelative effectiveness of the antisense sequences without regard toantisense chemistry or cell delivery. The relative location of thecertain exon 51-targeted oligos compared to SEQ ID NO: 327 is shown inFIG. 2D. As shown in FIG. 2C, there is a ranked hierarchy ofexon-skipping effectiveness, with SEQ ID NO: 327 being the mosteffective by at least a factor of several-fold compared to othersequences.

Example 2 Exon 50 Scan

A series of overlapping antisense PPMOs that target human dystrophinexon 50 were designed and synthesized. For exon 50, a series of 17PPMOs, each 25 bases in length, were made (SEQ ID NOS:267, 269, 271,273, 275, 277, 279, 280, 282 and 284-291) as shown in FIG. 3A. The PPMOswere evaluated for exon skipping efficacy by treating RD cells atvarious concentrations as described above in the Materials and Methods.Four PPMOs (SEQ ID NOS: 277, 287, 290 and 291) were identified aseffective in inducing exon-skipping and selected for additionalevaluation. Dose-ranging experiments in RD cells were used to confirmthe relative efficacy of these four PMO sequences. SEQ ID NOs: 584(AVI-5656) and 287 (AVI-5038) were shown to be most effective atinducing exon 50 skipping as shown in FIG. 3B. The EC₅₀ values werederived from the dose-ranging experiments and represent the calculatedconcentration where 50% of the PCR product is from the mRNA lacking exon50 relative to the PCR product produced from the mRNA containing exon50. Compared to other sequences (see, e.g., SEQ ID NOs: 584 and 585correspond to SEQ ID NOs: 173 and 175 in WO2006/000057, respectively)AVI-5038 (SEQ ID NO: 287) is equivalent or better at inducingexon-skipping activity in the RD cell assay as shown in FIG. 3B.

Example 3 Exon 53 Scan

A series of overlapping antisense PPMOs that target human dystrophinexon 53 were designed and synthesized. For exon 53, a series of 24PPMOs, each 25 bases in length, were made (SEQ ID NOS:416, 418, 420,422, 424, 426, 428, 429, 431, 433, 434, 436, 438-440 and 443-451) asshown in FIG. 4A. The PPMOs were evaluated for exon skipping efficacy bytreating RD cells and primary human skeletal muscle cells at variousconcentrations as described above in the Materials and Methods. ThreePPMOs (SEQ ID NOS: 428, 429 and 431) were identified as effective ininducing exon-skipping and selected for additional evaluation.Dose-ranging experiments in RD cells were used to confirm the relativeefficacy of these three PMO sequences. SEQ ID NO: 429 was shown to bemost effective at inducing exon 53 skipping as shown in FIGS. 4B-F.However, when compared to other exon 53 antisense sequences, SEQ ID NO:429 proved identical to H53A(+23+47) which is listed as SEQ ID NO: 195in WO2006/000057 and SEQ ID NO: 609 in the present application. Othersequences were compared to SEQ ID NO: 429 including H53A(+39+69) andH53A(−12+10) (listed as SEQ ID NOs:193 and 199 in WO2006/000057,respectively) and h53AON1 (listed as SEQ ID NO:39 in U.S. applicationSer. No. 11/233,507) and listed as SEQ ID NOs: 608, 611 and 610,respectively, in the present application. All the evaluated sequenceswere made as peptide-conjugated PMOs using the CP06062 peptide (SEQ IDNO: 578). This allowed direct comparison of the relative effectivenessof the antisense sequences without regard to antisense chemistry or celldelivery. As shown in FIGS. 4I and 4G-H, SEQ ID NO: 429 was shown to besuperior to each of these four sequences.

Example 4 Exon 44 Scan

A series of overlapping antisense PPMOs that target human dystrophinexon 44 were designed and synthesized. For exon 44, a series of PPMOs,each 25 bases in length, were made (SEQ ID NOS:1-20) as shown in FIG.5A. The PPMOs were evaluated for exon skipping efficacy by treating RDcells at various concentrations as described above in the Materials andMethods. Five PPMOs (SEQ ID NOS:4, 8, 11, 12 and 13) were identified aseffective in inducing exon-skipping and selected for additionalevaluation. Dose-ranging experiments in RD cells were used to confirmthe relative efficacy of these five PPMO sequences as shown in FIGS. 5Cto 5H. SEQ ID NOs: 8, 11 and 12 were shown to be most effective atinducing exon 44 skipping as shown in FIG. 5H with SEQ ID NO:12 provingthe most efficacious.

Comparison of SEQ ID NO: 12 to other exon 44 antisense sequences wasdone in both RD cells and human primary skeletal muscle cells. All theevaluated sequences were made as peptide-conjugated PMOs using theCP06062 peptide (SEQ ID NO: 578). This allowed direct comparison of therelative effectiveness of the antisense sequences without regard toantisense chemistry or cell delivery.

The alignment of the sequences (SEQ ID NOS: 600, 601, 602 and 603) withSEQ ID NOS: 4, 8, 11 and 12 is shown in FIG. 5B. SEQ ID NOS: 601 and 603are listed as SEQ ID NOS: 165 and 167 in WO2006/000057. SEQ ID NO:602 islisted in WO2004/083446 and as SEQ ID NO: 21 in U.S. application Ser.No. 11/233,507. SEQ ID NO:600 was published in 2007 (Wilton, Fall et al.2007). The comparison in RD cells showed that both SEQ ID NOS: 602 and603 were superior to SEQ ID NO:12 (FIG. 5I). However, as shown in FIG.5J, in human primary skeletal muscle cells SEQ ID NO:12 was superior(8.86% exon skipping) to SEQ ID NO:602 (6.42%). Similar experiments areperformed with SEQ ID NO:603.

Example 5 Exon 45 Scan

A series of overlapping antisense PPMOs that target human dystrophinexon 45 were designed and synthesized. For exon 45, a series of 22PPMOs, each 25 bases in length, were made (SEQ ID NOS: 21, 23, 25, 27,29, 31, 32, 34, 35, 37, 39, 41, 43 and 45-53) as shown in FIG. 6A. ThePPMOs were evaluated for exon skipping efficacy by treating RD cells andhuman primary skeletal muscle cells at various concentrations asdescribed above in the Materials and Methods. Five PPMOs (SEQ ID NOS:27,29, 34, and 39) were identified as effective in inducing exon-skippingand selected for additional evaluation. Dose-ranging experiments in RDcells were used to confirm the relative efficacy of these four PMOsequences as shown in FIGS. 6C-G and summarized in FIG. 6H. SEQ ID NO:49 was used as a negative control in these experiments. SEQ ID NOs: 29and 34 were shown to be most effective at inducing exon 45 skipping asshown in FIG. 6H.

Comparison of SEQ ID NO: 34 to other exon 45 antisense sequences wasdone in both RD cells and human primary skeletal muscle cells. All theevaluated sequences were made as peptide-conjugated PMOs using theCP06062 peptide (SEQ ID NO: 578). This allowed direct comparison of therelative effectiveness of the antisense sequences without regard toantisense chemistry or cell delivery. The alignment of the sequences(SEQ ID NOS: 604, 605, 606 and 607) with SEQ ID NOS: 27, 29, 34 and 39is shown in FIG. 6B. SEQ ID NOS: 604 and 607 are listed as SEQ ID NOS:211 and 207 in WO2006/000057, respectively. SEQ ID NOS:605 and 606 arelisted in U.S. application Ser. No. 11/233,507 as SEQ ID NOS: 23 and 1,respectively. The comparison in RD cells showed that SEQ ID NO: 34 wassuperior to all four sequences evaluated as shown in FIG. 6I. Testing ofthese compounds in different populations of human primary skeletalmuscle cells is performed as described above.

Sequence ID Listing

Sequences are shown using the nucleotide base symbols common for DNA: A,G, C and T. Other antisense chemistries such as 2′-O-methyl use U inplace of T. Any of the bases may be substituted with inosine (I)especially in stretches of three or more G residues.

SEQ Name Sequences ID NO. Oligomer Targeting Sequences (5′ to 3′):Hu.DMD.Exon44.25.001 CTGCAGGTAAAAGCATATGGATCAA   1 Hu.DMD.Exon44.25.002ATCGCCTGCAGGTAAAAGCATATGG   2 Hu.DMD.Exon44.25.003GTCAAATCGCCTGCAGGTAAAAGCA   3 Hu.DMD.Exon44.25.004GATCTGTCAAATCGCCTGCAGGTAA   4 Hu.DMD.Exon44.25.005CAACAGATCTGTCAAATCGCCTGCA   5 Hu.DMD.Exon44.25.006TTTCTCAACAGATCTGTCAAATCGC   6 Hu.DMD.Exon44.25.007CCATTTCTCAACAGATCTGTCAAAT   7 Hu.DMD.Exon44.25.008ATAATGAAAACGCCGCCATTTCTCA   8 Hu.DMD.Exon44.25.009AAATATCTTTATATCATAATGAAAA   9 Hu.DMD.Exon44.25.010TGTTAGCCACTGATTAAATATCTTT  10 Hu.DMD.Exon44.25.011AAACTGTTCAGCTTCTGTTAGCCAC  11 Hu.DMD.Exon44.25.012TTGTGTCTTTCTGAGAAACTGTTCA  12 Hu.DMD.Exon44.25.013CCAATTCTCAGGAATTTGTGTCTTT  13 Hu.DMD.Exon44.25.014GTATTTAGCATGTTCCCAATTCTCA  14 Hu.DMD.Exon44.25.015CTTAAGATACCATTTGTATTTAGCA  15 Hu.DMD.Exon44.25.016CTTACCTTAAGATACCATTTGTATT  16 Hu.DMD.Exon44.25.017AAAGACTTACCTTAAGATACCATTT  17 Hu.DMD.Exon44.25.018AAATCAAAGACTTACCTTAAGATAC  18 Hu.DMD.Exon44.25.019AAAACAAATCAAAGACTTACCTTAA  19 Hu.DMD.Exon44.25.020TCGAAAAAACAAATCAAAGACTTAC  20 Hu.DMD.Exon45.25.001CTGTAAGATACCAAAAAGGCAAAAC  21 Hu.DMD.Exon45.25.002CCTGTAAGATACCAAAAAGGCAAAA  22 Hu.DMD.Exon45.25.002.AGTTCCTGTAAGATACCAAAAAGGC  23 2 Hu.DMD.Exon45.25.003GAGTTCCTGTAAGATACCAAAAAGG  24 Hu.DMD.Exon45.25.003.CCTGGAGTTCCTGTAAGATACCAAA  25 2 Hu.DMD.Exon45.25.004TCCTGGAGTTCCTGTAAGATACCAA  26 Hu.DMD.Exon45.25.004.GCCATCCTGGAGTTCCTGTAAGATA  27 2 Hu.DMD.Exon45.25.005TGCCATCCTGGAGTTCCTGTAAGAT  28 Hu.DMD.Exon45.25.005.CCAATGCCATCCTGGAGTTCCTGTA  29 2 Hu.DMD.Exon45.25.006CCCAATGCCATCCTGGAGTTCCTGT  30 Hu.DMD.Exon45.25.006.GCTGCCCAATGCCATCCTGGAGTTC  31 2 Hu.DMD.Exon45.25.007CGCTGCCCAATGCCATCCTGGAGTT  32 Hu.DMD.Exon45.25.008AACAGTTTGCCGCTGCCCAATGCCA  33 Hu.DMD.Exon45.25.008.CTGACAACAGTTTGCCGCTGCCCAA  34 2 Hu.DMD.Exon45.25.009GTTGCATTCAATGTTCTGACAACAG  35 Hu.DMD.Exon45.25.010GCTGAATTATTTCTTCCCCAGTTGC  36 Hu.DMD.Exon45.25.010.ATTATTTCTTCCCCAGTTGCATTCA  37 2 Hu.DMD.Exon45.25.011GGCATCTGTTTTTGAGGATTGCTGA  38 Hu.DMD.Exon45.25.011.TTTGAGGATTGCTGAATTATTTCTT  39 2 Hu.DMD.Exon45.25.012AATTTTTCCTGTAGAATACTGGCAT  40 Hu.DMD.Exon45.25.012.ATACTGGCATCTGTTTTTGAGGATT  41 2 Hu.DMD.Exon45.25.013ACCGCAGATTCAGGCTTCCCAATTT  42 Hu.DMD.Exon45.25.013.AATTTTTCCTGTAGAATACTGGCAT  43 2 Hu.DMD.Exon45.25.014CTGTTTGCAGACCTCCTGCCACCGC  44 Hu.DMD.Exon45.25.014.AGATTCAGGCTTCCCAATTTTTCCT  45 2 Hu.DMD.Exon45.25.015CTCTTTTTTCTGTCTGACAGCTGTT  46 Hu.DMD.Exon45.25.015.ACCTCCTGCCACCGCAGATTCAGGC  47 2 Hu.DMD.Exon45.25.016CCTACCTCTTTTTTCTGTCTGACAG  48 Hu.DMD.Exon45.25.016.GACAGCTGTTTGCAGACCTCCTGCC  49 2 Hu.DMD.Exon45.25.017GTCGCCCTACCTCTTTTTTCTGTCT  50 Hu.DMD.Exon45.25.018GATCTGTCGCCCTACCTCTTTTTTC  51 Hu.DMD.Exon45.25.019TATTAGATCTGTCGCCCTACCTCTT  52 Hu.DMD.Exon45.25.020ATTCCTATTAGATCTGTCGCCCTAC  53 Hu.DMD.Exon45.20.001 AGATACCAAAAAGGCAAAAC 54 Hu.DMD.Exon45.20.002 AAGATACCAAAAAGGCAAAA  55 Hu.DMD.Exon45.20.003CCTGTAAGATACCAAAAAGG  56 Hu.DMD.Exon45.20.004 GAGTTCCTGTAAGATACCAA  57Hu.DMD.Exon45.20.005 TCCTGGAGTTCCTGTAAGAT  58 Hu.DMD.Exon45.20.006TGCCATCCTGGAGTTCCTGT  59 Hu.DMD.Exon45.20.007 CCCAATGCCATCCTGGAGTT  60Hu.DMD.Exon45.20.008 CGCTGCCCAATGCCATCCTG  61 Hu.DMD.Exon45.20.009CTGACAACAGTTTGCCGCTG  62 Hu.DMD.Exon45.20.010 GTTGCATTCAATGTTCTGAC  63Hu.DMD.Exon45.20.011 ATTATTTCTTCCCCAGTTGC  64 Hu.DMD.Exon45.20.012TTTGAGGATTGCTGAATTAT  65 Hu.DMD.Exon45.20.013 ATACTGGCATCTGTTTTTGA  66Hu.DMD.Exon45.20.014 AATTTTTCCTGTAGAATACT  67 Hu.DMD.Exon45.20.015AGATTCAGGCTTCCCAATTT  68 Hu.DMD.Exon45.20.016 ACCTCCTGCCACCGCAGATT  69Hu.DMD.Exon45.20.017 GACAGCTGTTTGCAGACCTC  70 Hu.DMD.Exon45.20.018CTCTTTTTTCTGTCTGACAG  71 Hu.DMD.Exon45.20.019 CCTACCTCTTTTTTCTGTCT  72Hu.DMD.Exon45.20.020 GTCGCCCTACCTCTTTTTTC  73 Hu.DMD.Exon45.20.021GATCTGTCGCCCTACCTCTT  74 Hu.DMD.Exon45.20.022 TATTAGATCTGTCGCCCTAC  75Hu.DMD.Exon45.20.023 ATTCCTATTAGATCTGTCGC  76 Hu.DMD.Exon46.25.001GGGGGATTTGAGAAAATAAAATTAC  77 Hu.DMD.Exon46.25.002ATTTGAGAAAATAAAATTACCTTGA  78 Hu.DMD.Exon46.25.002.CTAGCCTGGAGAAAGAAGAATAAAA  79 2 Hu.DMD.Exon46.25.003AGAAAATAAAATTACCTTGACTTGC  80 Hu.DMD.Exon46.25.003.TTCTTCTAGCCTGGAGAAAGAAGAA  81 2 Hu.DMD.Exon46.25.004ATAAAATTACCTTGACTTGCTCAAG  82 Hu.DMD.Exon46.25.004.TTTTGTTCTTCTAGCCTGGAGAAAG  83 2 Hu.DMD.Exon46.25.005ATTACCTTGACTTGCTCAAGCTTTT  84 Hu.DMD.Exon46.25.005.TATTCTTTTGTTCTTCTAGCCTGGA  85 2 Hu.DMD.Exon46.25.006CTTGACTTGCTCAAGCTTTTCTTTT  86 Hu.DMD.Exon46.25.006.CAAGATATTCTTTTGTTCTTCTAGC  87 2 Hu.DMD.Exon46.25.007CTTTTAGTTGCTGCTCTTTTCCAGG  88 Hu.DMD.Exon46.25.008CCAGGTTCAAGTGGGATACTAGCAA  89 Hu.DMD.Exon46.25.008.ATCTCTTTGAAATTCTGACAAGATA  90 2 Hu.DMD.Exon46.25.009AGCAATGTTATCTGCTTCCTCCAAC  91 Hu.DMD.Exon46.25.009.AACAAATTCATTTAAATCTCTTTGA  92 2 Hu.DMD.Exon46.25.010CCAACCATAAAACAAATTCATTTAA  93 Hu.DMD.Exon46.25.010.TTCCTCCAACCATAAAACAAATTCA  94 2 Hu.DMD.Exon46.25.011TTTAAATCTCTTTGAAATTCTGACA  95 Hu.DMD.Exon46.25.012TGACAAGATATTCTTTTGTTCTTCT  96 Hu.DMD.Exon46.25.012.TTCAAGTGGGATACTAGCAATGTTA  97 2 Hu.DMD.Exon46.25.013AGATATTCTTTTGTTCTTCTAGCCT  98 Hu.DMD.Exon46.25.013.CTGCTCTTTTCCAGGTTCAAGTGGG  99 2 Hu.DMD.Exon46.25.014TTCTTTTGTTCTTCTAGCCTGGAGA 100 Hu.DMD.Exon46.25.014.CTTTTCTTTTAGTTGCTGCTCTTTT 101 2 Hu.DMD.Exon46.25.015TTGTTCTTCTAGCCTGGAGAAAGAA 102 Hu.DMD.Exon46.25.016CTTCTAGCCTGGAGAAAGAAGAATA 103 Hu.DMD.Exon46.25.017AGCCTGGAGAAAGAAGAATAAAATT 104 Hu.DMD.Exon46.25.018CTGGAGAAAGAAGAATAAAATTGTT 105 Hu.DMD.Exon46.20.001 GAAAGAAGAATAAAATTGTT106 Hu.DMD.Exon46.20.002 GGAGAAAGAAGAATAAAATT 107 Hu.DMD.Exon46.20.003AGCCTGGAGAAAGAAGAATA 108 Hu.DMD.Exon46.20.004 CTTCTAGCCTGGAGAAAGAA 109Hu.DMD.Exon46.20.005 TTGTTCTTCTAGCCTGGAGA 110 Hu.DMD.Exon46.20.006TTCTTTTGTTCTTCTAGCCT 111 Hu.DMD.Exon46.20.007 TGACAAGATATTCTTTTGTT 112Hu.DMD.Exon46.20.008 ATCTCTTTGAAATTCTGACA 113 Hu.DMD.Exon46.20.009AACAAATTCATTTAAATCTC 114 Hu.DMD.Exon46.20.010 TTCCTCCAACCATAAAACAA 115Hu.DMD.Exon46.20.011 AGCAATGTTATCTGCTTCCT 116 Hu.DMD.Exon46.20.012TTCAAGTGGGATACTAGCAA 117 Hu.DMD.Exon46.20.013 CTGCTCTTTTCCAGGTTCAA 118Hu.DMD.Exon46.20.014 CTTTTCTTTTAGTTGCTGCT 119 Hu.DMD.Exon46.20.015CTTGACTTGCTCAAGCTTTT 120 Hu.DMD.Exon46.20.016 ATTACCTTGACTTGCTCAAG 121Hu.DMD.Exon46.20.017 ATAAAATTACCTTGACTTGC 122 Hu.DMD.Exon46.20.018AGAAAATAAAATTACCTTGA 123 Hu.DMD.Exon46.20.019 ATTTGAGAAAATAAAATTAC 124Hu.DMD.Exon46.20.020 GGGGGATTTGAGAAAATAAA 125 Hu.DMD.Exon47.25.001CTGAAACAGACAAATGCAACAACGT 126 Hu.DMD.Exon47.25.002AGTAACTGAAACAGACAAATGCAAC 127 Hu.DMD.Exon47.25.003CCACCAGTAACTGAAACAGACAAAT 128 Hu.DMD.Exon47.25.004CTCTTCCACCAGTAACTGAAACAGA 129 Hu.DMD.Exon47.25.005GGCAACTCTTCCACCAGTAACTGAA 130 Hu.DMD.Exon47.25.006GCAGGGGCAACTCTTCCACCAGTAA 131 Hu.DMD.Exon47.25.007CTGGCGCAGGGGCAACTCTTCCACC 132 Hu.DMD.Exon47.25.008TTTAATTGTTTGAGAATTCCCTGGC 133 Hu.DMD.Exon47.25.008.TTGTTTGAGAATTCCCTGGCGCAGG 134 2 Hu.DMD.Exon47.25.009GCACGGGTCCTCCAGTTTCATTTAA 135 Hu.DMD.Exon47.25.009.TCCAGTTTCATTTAATTGTTTGAGA 136 2 Hu.DMD.Exon47.25.010GCTTATGGGAGCACTTACAAGCACG 137 Hu.DMD.Exon47.25.010.TACAAGCACGGGTCCTCCAGTTTCA 138 2 Hu.DMD.Exon47.25.011AGTTTATCTTGCTCTTCTGGGCTTA 139 Hu.DMD.Exon47.25.012TCTGCTTGAGCTTATTTTCAAGTTT 140 Hu.DMD.Exon47.25.012.ATCTTGCTCTTCTGGGCTTATGGGA 141 2 Hu.DMD.Exon47.25.013CTTTATCCACTGGAGATTTGTCTGC 142 Hu.DMD.Exon47.25.013.CTTATTTTCAAGTTTATCTTGCTCT 143 2 Hu.DMD.Exon47.25.014CTAACCTTTATCCACTGGAGATTTG 144 Hu.DMD.Exon47.25.014.ATTTGTCTGCTTGAGCTTATTTTCA 145 2 Hu.DMD.Exon47.25.015AATGTCTAACCTTTATCCACTGGAG 146 Hu.DMD.Exon47.25.016TGGTTAATGTCTAACCTTTATCCAC 147 Hu.DMD.Exon47.25.017AGAGATGGTTAATGTCTAACCTTTA 148 Hu.DMD.Exon47.25.018ACGGAAGAGATGGTTAATGTCTAAC 149 Hu.DMD.Exon47.20.001 ACAGACAAATGCAACAACGT150 Hu.DMD.Exon47.20.002 CTGAAACAGACAAATGCAAC 151 Hu.DMD.Exon47.20.003AGTAACTGAAACAGACAAAT 152 Hu.DMD.Exon47.20.004 CCACCAGTAACTGAAACAGA 153Hu.DMD.Exon47.20.005 CTCTTCCACCAGTAACTGAA 154 Hu.DMD.Exon47.20.006GGCAACTCTTCCACCAGTAA 155 Hu.DMD.Exon47.20.007 CTGGCGCAGGGGCAACTCTT 156Hu.DMD.Exon47.20.008 TTGTTTGAGAATTCCCTGGC 157 Hu.DMD.Exon47.20.009TCCAGTTTCATTTAATTGTT 158 Hu.DMD.Exon47.20.010 TACAAGCACGGGTCCTCCAG 159Hu.DMD.Exon47.20.011 GCTTATGGGAGCACTTACAA 160 Hu.DMD.Exon47.20.012ATCTTGCTCTTCTGGGCTTA 161 Hu.DMD.Exon47.20.013 CTTATTTTCAAGTTTATCTT 162Hu.DMD.Exon47.20.014 ATTTGTCTGCTTGAGCTTAT 163 Hu.DMD.Exon47.20.015CTTTATCCACTGGAGATTTG 164 Hu.DMD.Exon47.20.016 CTAACCTTTATCCACTGGAG 165Hu.DMD.Exon47.20.017 AATGTCTAACCTTTATCCAC 166 Hu.DMD.Exon47.20.018TGGTTAATGTCTAACCTTTA 167 Hu.DMD.Exon47.20.019 AGAGATGGTTAATGTCTAAC 168Hu.DMD.Exon47.20.020 ACGGAAGAGATGGTTAATGT 169 Hu.DMD.Exon48.25.001CTGAAAGGAAAATACATTTTAAAAA 170 Hu.DMD.Exon48.25.002CCTGAAAGGAAAATACATTTTAAAA 171 Hu.DMD.Exon48.25.002.GAAACCTGAAAGGAAAATACATTTT 172 2 Hu.DMD.Exon48.25.003GGAAACCTGAAAGGAAAATACATTT 173 Hu.DMD.Exon48.25.003.CTCTGGAAACCTGAAAGGAAAATAC 174 2 Hu.DMD.Exon48.25.004GCTCTGGAAACCTGAAAGGAAAATA 175 Hu.DMD.Exon48.25.004.TAAAGCTCTGGAAACCTGAAAGGAA 634 2 Hu.DMD.Exon48.25.005GTAAAGCTCTGGAAACCTGAAAGGA 176 Hu.DMD.Exon48.25.005.TCAGGTAAAGCTCTGGAAACCTGAA 177 2 Hu.DMD.Exon48.25.006CTCAGGTAAAGCTCTGGAAACCTGA 178 Hu.DMD.Exon48.25.006.GTTTCTCAGGTAAAGCTCTGGAAAC 179 2 Hu.DMD.Exon48.25.007TGTTTCTCAGGTAAAGCTCTGGAAA 180 Hu.DMD.Exon48.25.007.AATTTCTCCTTGTTTCTCAGGTAAA 181 2 Hu.DMD.Exon48.25.008TTTGAGCTTCAATTTCTCCTTGTTT 182 Hu.DMD.Exon48.25.008TTTTATTTGAGCTTCAATTTCTCCT 183 Hu.DMD.Exon48.25.009AAGCTGCCCAAGGTCTTTTATTTGA 184 Hu.DMD.Exon48.25.010AGGTCTTCAAGCTTTTTTTCAAGCT 185 Hu.DMD.Exon48.25.010.TTCAAGCTTTTTTTCAAGCTGCCCA 186 2 Hu.DMD.Exon48.25.011GATGATTTAACTGCTCTTCAAGGTC 187 Hu.DMD.Exon48.25.011.CTGCTCTTCAAGGTCTTCAAGCTTT 188 2 Hu.DMD.Exon48.25.012AGGAGATAACCACAGCAGCAGATGA 189 Hu.DMD.Exon48.25.012.CAGCAGATGATTTAACTGCTCTTCA 190 2 Hu.DMD.Exon48.25.013ATTTCCAACTGATTCCTAATAGGAG 191 Hu.DMD.Exon48.25.014CTTGGTTTGGTTGGTTATAAATTTC 192 Hu.DMD.Exon48.25.014.CAACTGATTCCTAATAGGAGATAAC 193 2 Hu.DMD.Exon48.25.015CTTAACGTCAAATGGTCCTTCTTGG 194 Hu.DMD.Exon48.25.015.TTGGTTATAAATTTCCAACTGATTC 195 2 Hu.DMD.Exon48.25.016CCTACCTTAACGTCAAATGGTCCTT 196 Hu.DMD.Exon48.25.016.TCCTTCTTGGTTTGGTTGGTTATAA 197 2 Hu.DMD.Exon48.25.017AGTTCCCTACCTTAACGTCAAATGG 198 Hu.DMD.Exon48.25.018CAAAAAGTTCCCTACCTTAACGTCA 199 Hu.DMD.Exon48.25.019TAAAGCAAAAAGTTCCCTACCTTAA 200 Hu.DMD.Exon48.25.020ATATTTAAAGCAAAAAGTTCCCTAC 201 Hu.DMD.Exon48.20.001 AGGAAAATACATTTTAAAAA202 Hu.DMD.Exon48.20.002 AAGGAAAATACATTTTAAAA 203 Hu.DMD.Exon48.20.003CCTGAAAGGAAAATACATTT 204 Hu.DMD.Exon48.20.004 GGAAACCTGAAAGGAAAATA 205Hu.DMD.Exon48.20.005 GCTCTGGAAACCTGAAAGGA 206 Hu.DMD.Exon48.20.006GTAAAGCTCTGGAAACCTGA 207 Hu.DMD.Exon48.20.007 CTCAGGTAAAGCTCTGGAAA 208Hu.DMD.Exon48.20.008 AATTTCTCCTTGTTTCTCAG 209 Hu.DMD.Exon48.20.009TTTTATTTGAGCTTCAATTT 210 Hu.DMD.Exon48.20.010 AAGCTGCCCAAGGTCTTTTA 211Hu.DMD.Exon48.20.011 TTCAAGCTTTTTTTCAAGCT 212 Hu.DMD.Exon48.20.012CTGCTCTTCAAGGTCTTCAA 213 Hu.DMD.Exon48.20.013 CAGCAGATGATTTAACTGCT 214Hu.DMD.Exon48.20.014 AGGAGATAACCACAGCAGCA 215 Hu.DMD.Exon48.20.015CAACTGATTCCTAATAGGAG 216 Hu.DMD.Exon48.20.016 TTGGTTATAAATTTCCAACT 217Hu.DMD.Exon48.20.017 TCCTTCTTGGTTTGGTTGGT 218 Hu.DMD.Exon48.20.018CTTAACGTCAAATGGTCCTT 219 Hu.DMD.Exon48.20.019 CCTACCTTAACGTCAAATGG 220Hu.DMD.Exon48.20.020 AGTTCCCTACCTTAACGTCA 221 Hu.DMD.Exon48.20.021CAAAAAGTTCCCTACCTTAA 222 Hu.DMD.Exon48.20.022 TAAAGCAAAAAGTTCCCTAC 223Hu.DMD.Exon48.20.023 ATATTTAAAGCAAAAAGTTC 224 Hu.DMD.Exon49.25.001CTGGGGAAAAGAACCCATATAGTGC 225 Hu.DMD.Exon49.25.002TCCTGGGGAAAAGAACCCATATAGT 226 Hu.DMD.Exon49.25.002.GTTTCCTGGGGAAAAGAACCCATAT 227 2 Hu.DMD.Exon49.25.003CAGTTTCCTGGGGAAAAGAACCCAT 228 Hu.DMD.Exon49.25.003.TTTCAGTTTCCTGGGGAAAAGAACC 229 2 Hu.DMD.Exon49.25.004TATTTCAGTTTCCTGGGGAAAAGAA 230 Hu.DMD.Exon49.25.004.TGCTATTTCAGTTTCCTGGGGAAAA 231 2 Hu.DMD.Exon49.25.005ACTGCTATTTCAGTTTCCTGGGGAA 232 Hu.DMD.Exon49.25.005.TGAACTGCTATTTCAGTTTCCTGGG 233 2 Hu.DMD.Exon49.25.006CTTGAACTGCTATTTCAGTTTCCTG 234 Hu.DMD.Exon49.25.006.TAGCTTGAACTGCTATTTCAGTTTC 235 2 Hu.DMD.Exon49.25.007TTTAGCTTGAACTGCTATTTCAGTT 236 Hu.DMD.Exon49.25.008TTCCACATCCGGTTGTTTAGCTTGA 237 Hu.DMD.Exon49.25.009TGCCCTTTAGACAAAATCTCTTCCA 238 Hu.DMD.Exon49.25.009.TTTAGACAAAATCTCTTCCACATCC 239 2 Hu.DMD.Exon49.25.010GTTTTTCCTTGTACAAATGCTGCCC 240 Hu.DMD.Exon49.25.010.GTACAAATGCTGCCCTTTAGACAAA 241 2 Hu.DMD.Exon49.25.011CTTCACTGGCTGAGTGGCTGGTTTT 242 Hu.DMD.Exon49.25.011.GGCTGGTTTTTCCTTGTACAAATGC 243 2 Hu.DMD.Exon49.25.012ATTACCTTCACTGGCTGAGTGGCTG 244 Hu.DMD.Exon49.25.013GCTTCATTACCTTCACTGGCTGAGT 245 Hu.DMD.Exon49.25.014AGGTTGCTTCATTACCTTCACTGGC 246 Hu.DMD.Exon49.25.015GCTAGAGGTTGCTTCATTACCTTCA 247 Hu.DMD.Exon49.25.016ATATTGCTAGAGGTTGCTTCATTAC 248 Hu.DMD.Exon49.20.001 GAAAAGAACCCATATAGTGC249 Hu.DMD.Exon49.20.002 GGGAAAAGAACCCATATAGT 250 Hu.DMD.Exon49.20.003TCCTGGGGAAAAGAACCCAT 251 Hu.DMD.Exon49.20.004 CAGTTTCCTGGGGAAAAGAA 252Hu.DMD.Exon49.20.005 TATTTCAGTTTCCTGGGGAA 253 Hu.DMD.Exon49.20.006ACTGCTATTTCAGTTTCCTG 254 Hu.DMD.Exon49.20.007 CTTGAACTGCTATTTCAGTT 255Hu.DMD.Exon49.20.008 TTTAGCTTGAACTGCTATTT 256 Hu.DMD.Exon49.20.009TTCCACATCCGGTTGTTTAG 257 Hu.DMD.Exon49.20.010 TTTAGACAAAATCTCTTCCA 258Hu.DMD.Exon49.20.011 GTACAAATGCTGCCCTTTAG 259 Hu.DMD.Exon49.20.012GGCTGGTTTTTCCTTGTACA 260 Hu.DMD.Exon49.20.013 CTTCACTGGCTGAGTGGCTG 261Hu.DMD.Exon49.20.014 ATTACCTTCACTGGCTGAGT 262 Hu.DMD.Exon49.20.015GCTTCATTACCTTCACTGGC 263 Hu.DMD.Exon49.20.016 AGGTTGCTTCATTACCTTCA 264Hu.DMD.Exon49.20.017 GCTAGAGGTTGCTTCATTAC 265 Hu.DMD.Exon49.20.018ATATTGCTAGAGGTTGCTTC 266 Hu.DMD.Exon50.25.001 CTTTAACAGAAAAGCATACACATTA267 Hu.DMD.Exon50.25.002 TCCTCTTTAACAGAAAAGCATACAC 268Hu.DMD.Exon50.25.002. TTCCTCTTTAACAGAAAAGCATACA 269 2Hu.DMD.Exon50.25.003 TAACTTCCTCTTTAACAGAAAAGCA 270 Hu.DMD.Exon50.25.003.CTAACTTCCTCTTTAACAGAAAAGC 271 2 Hu.DMD.Exon50.25.004TCTTCTAACTTCCTCTTTAACAGAA 272 Hu.DMD.Exon50.25.004.ATCTTCTAACTTCCTCTTTAACAGA 273 2 Hu.DMD.Exon50.25.005TCAGATCTTCTAACTTCCTCTTTAA 274 Hu.DMD.Exon50.25.005.CTCAGATCTTCTAACTTCCTCTTTA 275 2 Hu.DMD.Exon50.25.006AGAGCTCAGATCTTCTAACTTCCTC 276 Hu.DMD.Exon50.25.006.CAGAGCTCAGATCTTCTAACTTCCT 277 2 NG-08-0731 Hu.DMD.Exon50.25.007CACTCAGAGCTCAGATCTTCTACT 278 Hu.DMD.Exon50.25.007.CCTTCCACTCAGAGCTCAGATCTTC 279 2 Hu.DMD.Exon50.25.008GTAAACGGTTTACCGCCTTCCACTC 280 Hu.DMD.Exon50.25.009CTTTGCCCTCAGCTCTTGAAGTAAA 281 Hu.DMD.Exon50.25.009.CCCTCAGCTCTTGAAGTAAACGGTT 282 2 Hu.DMD.Exon50.25.010CCAGGAGCTAGGTCAGGCTGCTTTG 283 Hu.DMD.Exon50.25.010.GGTCAGGCTGCTTTGCCCTCAGCTC 284 2 Hu.DMD.Exon50.25.011AGGCTCCAATAGTGGTCAGTCCAGG 285 Hu.DMD.Exon50.25.011.TCAGTCCAGGAGCTAGGTCAGGCTG 286 2 Hu.DMD.Exon50.25.012CTTACAGGCTCCAATAGTGGTCAGT 287 AVI-5038 Hu.DMD.Exon50.25.013GTATACTTACAGGCTCCAATAGTGG 288 Hu.DMD.Exon50.25.014ATCCAGTATACTTACAGGCTCCAAT 289 Hu.DMD.Exon50.25.015ATGGGATCCAGTATACTTACAGGCT 290 NG-08-0741 Hu.DMD.Exon50.25.016AGAGAATGGGATCCAGTATACTTAC 291 NG-08-0742 Hu.DMD.Exon50.20.001ACAGAAAAGCATACACATTA 292 Hu.DMD.Exon50.20.002 TTTAACAGAAAAGCATACAC 293Hu.DMD.Exon50.20.003 TCCTCTTTAACAGAAAAGCA 294 Hu.DMD.Exon50.20.004TAACTTCCTCTTTAACAGAA 295 Hu.DMD.Exon50.20.005 TCTTCTAACTTCCTCTTTAA 296Hu.DMD.Exon50.20.006 TCAGATCTTCTAACTTCCTC 297 Hu.DMD.Exon50.20.007CCTTCCACTCAGAGCTCAGA 298 Hu.DMD.Exon50.20.008 GTAAACGGTTTACCGCCTTC 299Hu.DMD.Exon50.20.009 CCCTCAGCTCTTGAAGTAAA 300 Hu.DMD.Exon50.20.010GGTCAGGCTGCTTTGCCCTC 301 Hu.DMD.Exon50.20.011 TCAGTCCAGGAGCTAGGTCA 302Hu.DMD.Exon50.20.012 AGGCTCCAATAGTGGTCAGT 303 Hu.DMD.Exon50.20.013CTTACAGGCTCCAATAGTGG 304 Hu.DMD.Exon50.20.014 GTATACTTACAGGCTCCAAT 305Hu.DMD.Exon50.20.015 ATCCAGTATACTTACAGGCT 306 Hu.DMD.Exon50.20.016ATGGGATCCAGTATACTTAC 307 Hu.DMD.Exon50.20.017 AGAGAATGGGATCCAGTATA 308Hu.DMD.Exon51.25.001- CTAAAATATTTTGGGTTTTTGCAAAA 309 44Hu.DMD.Exon51.25.002- GCTAAAATATTTTGGGTTTTTGCAAA 310 45Hu.DMD.Exon51.25.002. TAGGAGCTAAAATATTTTGGGTTTTT 311 2-46Hu.DMD.Exon51.25.003 AGTAGGAGCTAAAATATTTTGGGTT 312 Hu.DMD.Exon51.25.003.TGAGTAGGAGCTAAAATATTTTGGG 313 2 Hu.DMD.Exon51.25.004CTGAGTAGGAGCTAAAATATTTTGGG 314 Hu.DMD.Exon51.25.004.CAGTCTGAGTAGGAGCTAAAATATT 315 2 Hu.DMD.Exon51.25.005ACAGTCTGAGTAGGAGCTAAAATATT 316 Hu.DMD.Exon51.25.005.GAGTAACAGTCTGAGTAGGAGCTAAA 317 2 Hu.DMD.Exon51.25.006CAGAGTAACAGTCTGAGTAGGAGCT 318 Hu.DMD.Exon51.25.006.CACCAGAGTAACAGTCTGAGTAGGAG 319 2 Hu.DMD.Exon51.25.007GTCACCAGAGTAACAGTCTGAGTAG 320 Hu.DMD.Exon51.25.007.AACCACAGGTTGTGTCACCAGAGTAA 321 2 Hu.DMD.Exon51.25.008GTTGTGTCACCAGAGTAACAGTCTG 322 Hu.DMD.Exon51.25.009TGGCAGTTTCCTTAGTAACCACAGGT 323 Hu.DMD.Exon51.25.010ATTTCTAGTTTGGAGATGGCAGTTTC 324 Hu.DMD.Exon51.25.010.GGAAGATGGCATTTCTAGTTTGGAG 325 2 Hu.DMD.Exon51.25.011CATCAAGGAAGATGGCATTTCTAGTT 326 Hu.DMD.Exon51.25.011.GAGCAGGTACCTCCAACATCAAGGAA 327 2 Hu.DMD.Exon51.25.012ATCTGCCAGAGCAGGTACCTCCAAC 328 Hu.DMD.Exon51.25.013AAGTTCTGTCCAAGCCCGGTTGAAAT 329 Hu.DMD.Exon51.25.013.CGGTTGAAATCTGCCAGAGCAGGTAC 330 2 Hu.DMD.Exon51.25.014GAGAAAGCCAGTCGGTAAGTTCTGTC 331 Hu.DMD.Exon51.25.014.GTCGGTAAGTTCTGTCCAAGCCCGG 332 2 Hu.DMD.Exon51.25.015ATAACTTGATCAAGCAGAGAAAGCCA 333 Hu.DMD.Exon51.25.015.AAGCAGAGAAAGCCAGTCGGTAAGT 334 2 Hu.DMD.Exon51.25.016CACCCTCTGTGATTTTATAACTTGAT 335 Hu.DMD.Exon51.25.017CAAGGTCACCCACCATCACCCTCTGT 336 Hu.DMD.Exon51.25.017.CATCACCCTCTGTGATTTTATAACT 337 2 Hu.DMD.Exon51.25.018CTTCTGCTTGATGATCATCTCGTTGA 338 Hu.DMD.Exon51.25.019CCTTCTGCTTGATGATCATCTCGTTG 339 Hu.DMD.Exon51.25.019.ATCTCGTTGATATCCTCAAGGTCACC 340 2 Hu.DMD.Exon51.25.020TCATACCTTCTGCTTGATGATCATCT 341 Hu.DMD.Exon51.25.020.TCATTTTTTCTCATACCTTCTGCTTG 342 2 Hu.DMD.Exon51.25.021TTTTCTCATACCTTCTGCTTGATGAT 343 Hu.DMD.Exon51.25.022TTTTATCATTTTTTCTCATACCTTCT 344 Hu.DMD.Exon51.25.023CCAACTTTTATCATTTTTTCTCATAC 345 Hu.DMD.Exon51.20.001 ATATTTTGGGTTTTTGCAAA346 Hu.DMD.Exon51.20.002 AAAATATTTTGGGTTTTTGC 347 Hu.DMD.Exon51.20.003GAGCTAAAATATTTTGGGTT 348 Hu.DMD.Exon51.20.004 AGTAGGAGCTAAAATATTTT 349Hu.DMD.Exon51.20.005 GTCTGAGTAGGAGCTAAAAT 350 Hu.DMD.Exon51.20.006TAACAGTCTGAGTAGGAGCT 351 Hu.DMD.Exon51.20.007 CAGAGTAACAGTCTGAGTAG 352Hu.DMD.Exon51.20.008 CACAGGTTGTGTCACCAGAG 353 Hu.DMD.Exon51.20.009AGTTTCCTTAGTAACCACAG 354 Hu.DMD.Exon51.20.010 TAGTTTGGAGATGGCAGTTT 355Hu.DMD.Exon51.20.011 GGAAGATGGCATTTCTAGTT 356 Hu.DMD.Exon51.20.012TACCTCCAACATCAAGGAAG 357 Hu.DMD.Exon51.20.013 ATCTGCCAGAGCAGGTACCT 358Hu.DMD.Exon51.20.014 CCAAGCCCGGTTGAAATCTG 359 Hu.DMD.Exon51.20.015GTCGGTAAGTTCTGTCCAAG 360 Hu.DMD.Exon51.20.016 AAGCAGAGAAAGCCAGTCGG 361Hu.DMD.Exon51.20.017 TTTTATAACTTGATCAAGCA 362 Hu.DMD.Exon51.20.018CATCACCCTCTGTGATTTTA 363 Hu.DMD.Exon51.20.019 CTCAAGGTCACCCACCATCA 364Hu.DMD.Exon51.20.020 CATCTCGTTGATATCCTCAA 365 Hu.DMD.Exon51.20.021CTTCTGCTTGATGATCATCT 366 Hu.DMD.Exon51.20.022 CATACCTTCTGCTTGATGAT 367Hu.DMD.Exon51.20.023 TTTCTCATACCTTCTGCTTG 368 Hu.DMD.Exon51.20.024CATTTTTTCTCATACCTTCT 369 Hu.DMD.Exon51.20.025 TTTATCATTTTTTCTCATAC 370Hu.DMD.Exon51.20.026 CAACTTTTATCATTTTTTCT 371 Hu.DMD.Exon52.25.001CTGTAAGAACAAATATCCCTTAGTA 372 Hu.DMD.Exon52.25.002TGCCTGTAAGAACAAATATCCCTTA 373 Hu.DMD.Exon52.25.002.GTTGCCTGTAAGAACAAATATCCCT 374 2 Hu.DMD.Exon52.25.003ATTGTTGCCTGTAAGAACAAATATC 375 Hu.DMD.Exon52.25.003.GCATTGTTGCCTGTAAGAACAAATA 376 2 Hu.DMD.Exon52.25.004CCTGCATTGTTGCCTGTAAGAACAA 377 Hu.DMD.Exon52.25.004.ATCCTGCATTGTTGCCTGTAAGAAC 378 2 Hu.DMD.Exon52.25.005CAAATCCTGCATTGTTGCCTGTAAG 379 Hu.DMD.Exon52.25.005.TCCAAATCCTGCATTGTTGCCTGTA 380 2 Hu.DMD.Exon52.25.006TGTTCCAAATCCTGCATTGTTGCCT 381 Hu.DMD.Exon52.25.006.TCTGTTCCAAATCCTGCATTGTTGC 382 2 Hu.DMD.Exon52.25.007AACTGGGGACGCCTCTGTTCCAAAT 383 Hu.DMD.Exon52.25.007.GCCTCTGTTCCAAATCCTGCATTGT 384 2 Hu.DMD.Exon52.25.008CAGCGGTAATGAGTTCTTCCAACTG 385 Hu.DMD.Exon52.25.008.CTTCCAACTGGGGACGCCTCTGTTC 386 2 Hu.DMD.Exon52.25.009CTTGTTTTTCAAATTTTGGGCAGCG 387 Hu.DMD.Exon52.25.010CTAGCCTCTTGATTGCTGGTCTTGT 388 Hu.DMD.Exon52.25.010.TTTTCAAATTTTGGGCAGCGGTAAT 389 2 Hu.DMD.Exon52.25.011TTCGATCCGTAATGATTGTTCTAGC 390 Hu.DMD.Exon52.25.011.GATTGCTGGTCTTGTTTTTCAAATT 391 2 Hu.DMD.Exon52.25.012CTTACTTCGATCCGTAATGATTGTT 392 Hu.DMD.Exon52.25.012.TTGTTCTAGCCTCTTGATTGCTGGT 393 2 Hu.DMD.Exon52.25.013AAAAACTTACTTCGATCCGTAATGA 394 Hu.DMD.Exon52.25.014TGTTAAAAAACTTACTTCGATCCGT 395 Hu.DMD.Exon52.25.015ATGCTTGTTAAAAAACTTACTTCGA 396 Hu.DMD.Exon52.25.016GTCCCATGCTTGTTAAAAAACTTAC 397 Hu.DMD.Exon52.20.001 AGAACAAATATCCCTTAGTA398 Hu.DMD.Exon52.20.002 GTAAGAACAAATATCCCTTA 399 Hu.DMD.Exon52.20.003TGCCTGTAAGAACAAATATC 400 Hu.DMD.Exon52.20.004 ATTGTTGCCTGTAAGAACAA 401Hu.DMD.Exon52.20.005 CCTGCATTGTTGCCTGTAAG 402 Hu.DMD.Exon52.20.006CAAATCCTGCATTGTTGCCT 403 Hu.DMD.Exon52.20.007 GCCTCTGTTCCAAATCCTGC 404Hu.DMD.Exon52.20.008 CTTCCAACTGGGGACGCCTC 405 Hu.DMD.Exon52.20.009CAGCGGTAATGAGTTCTTCC 406 Hu.DMD.Exon52.20.010 TTTTCAAATTTTGGGCAGCG 407Hu.DMD.Exon52.20.011 GATTGCTGGTCTTGTTTTTC 408 Hu.DMD.Exon52.20.012TTGTTCTAGCCTCTTGATTG 409 Hu.DMD.Exon52.20.013 TTCGATCCGTAATGATTGTT 410Hu.DMD.Exon52.20.014 CTTACTTCGATCCGTAATGA 411 Hu.DMD.Exon52.20.015AAAAACTTACTTCGATCCGT 412 Hu.DMD.Exon52.20.016 TGTTAAAAAACTTACTTCGA 413Hu.DMD.Exon52.20.017 ATGCTTGTTAAAAAACTTAC 414 Hu.DMD.Exon52.20.018GTCCCATGCTTGTTAAAAAA 415 Hu.DMD.Exon53.25.001 CTAGAATAAAAGGAAAAATAAATAT416 Hu.DMD.Exon53.25.002 AACTAGAATAAAAGGAAAAATAAAT 417Hu.DMD.Exon53.25.002. TTCAACTAGAATAAAAGGAAAAATA 418 2Hu.DMD.Exon53.25.003 CTTTCAACTAGAATAAAAGGAAAAA 419 Hu.DMD.Exon53.25.003.ATTCTTTCAACTAGAATAAAAGGAA 420 2 Hu.DMD.Exon53.25.004GAATTCTTTCAACTAGAATAAAAGG 421 Hu.DMD.Exon53.25.004.TCTGAATTCTTTCAACTAGAATAAA 422 2 Hu.DMD.Exon53.25.005ATTCTGAATTCTTTCAACTAGAATA 423 Hu.DMD.Exon53.25.005.CTGATTCTGAATTCTTTCAACTAGA 424 2 Hu.DMD.Exon53.25.006CACTGATTCTGAATTCTTTCAACTA 425 Hu.DMD.Exon53.25.006.TCCCACTGATTCTGAATTCTTTCAA 426 2 Hu.DMD.Exon53.25.007CATCCCACTGATTCTGAATTCTTTC 427 Hu.DMD.Exon53.25.008TACTTCATCCCACTGATTCTGAATT 428 Hu.DMD.Exon53.25.008.CTGAAGGTGTTCTTGTACTTCATCC 429 2 Hu.DMD.Exon53.25.009CGGTTCTGAAGGTGTTCTTGTACT 430 Hu.DMD.Exon53.25.009.CTGTTGCCTCCGGTTCTGAAGGTGT 431 2 Hu.DMD.Exon53.25.010TTTCATTCAACTGTTGCCTCCGGTT 432 Hu.DMD.Exon53.25.010.TAACATTTCATTCAACTGTTGCCTC 433 2 Hu.DMD.Exon53.25.011TTGTGTTGAATCCTTTAACATTTCA 434 Hu.DMD.Exon53.25.012TCTTCCTTAGCTTCCAGCCATTGTG 435 Hu.DMD.Exon53.25.012.CTTAGCTTCCAGCCATTGTGTTGAA 436 2 Hu.DMD.Exon53.25.013GTCCTAAGACCTGCTCAGCTTCTTC 437 Hu.DMD.Exon53.25.013.CTGCTCAGCTTCTTCCTTAGCTTCC 438 2 Hu.DMD.Exon53.25.014CTCAAGCTTGGCTCTGGCCTGTCCT 439 Hu.DMD.Exon53.25.014.GGCCTGTCCTAAGACCTGCTCAGCT 440 2 Hu.DMD.Exon53.25.015TAGGGACCCTCCTTCCATGACTCAA 441 Hu.DMD.Exon53.25.016TTTGGATTGCATCTACTGTATAGGG 442 Hu.DMD.Exon53.25.016.ACCCTCCTTCCATGACTCAAGCTTG 443 2 Hu.DMD.Exon53.25.017CTTGGTTTCTGTGATTTTCTTTTGG 444 Hu.DMD.Exon53.25.017.ATCTACTGTATAGGGACCCTCCTTC 445 2 Hu.DMD.Exon53.25.018CTAACCTTGGTTTCTGTGATTTTCT 446 Hu.DMD.Exon53.25.018.TTTCTTTTGGATTGCATCTACTGTA 447 2 Hu.DMD.Exon53.25.019TGATACTAACCTTGGTTTCTGTGAT 448 Hu.DMD.Exon53.25.020ATCTTTGATACTAACCTTGGTTTCT 449 Hu.DMD.Exon53.25.021AAGGTATCTTTGATACTAACCTTGG 450 Hu.DMD.Exon53.25.022TTAAAAAGGTATCTTTGATACTAAC 451 Hu.DMD.Exon53.20.001 ATAAAAGGAAAAATAAATAT452 Hu.DMD.Exon53.20.002 GAATAAAAGGAAAAATAAAT 453 Hu.DMD.Exon53.20.003AACTAGAATAAAAGGAAAAA 454 Hu.DMD.Exon53.20.004 CTTTCAACTAGAATAAAAGG 455Hu.DMD.Exon53.20.005 GAATTCTTTCAACTAGAATA 456 Hu.DMD.Exon53.20.006ATTCTGAATTCTTTCAACTA 457 Hu.DMD.Exon53.20.007 TACTTCATCCCACTGATTCT 458Hu.DMD.Exon53.20.008 CTGAAGGTGTTCTTGTACT 459 Hu.DMD.Exon53.20.009CTGTTGCCTCCGGTTCTGAA 460 Hu.DMD.Exon53.20.010 TAACATTTCATTCAACTGTT 461Hu.DMD.Exon53.20.011 TTGTGTTGAATCCTTTAACA 462 Hu.DMD.Exon53.20.012CTTAGCTTCCAGCCATTGTG 463 Hu.DMD.Exon53.20.013 CTGCTCAGCTTCTTCCTTAG 464Hu.DMD.Exon53.20.014 GGCCTGTCCTAAGACCTGCT 465 Hu.DMD.Exon53.20.015CTCAAGCTTGGCTCTGGCCT 466 Hu.DMD.Exon53.20.016 ACCCTCCTTCCATGACTCAA 467Hu.DMD.Exon53.20.017 ATCTACTGTATAGGGACCCT 468 Hu.DMD.Exon53.20.018TTTCTTTTGGATTGCATCTA 469 Hu.DMD.Exon53.20.019 CTTGGTTTCTGTGATTTTCT 470Hu.DMD.Exon53.20.020 CTAACCTTGGTTTCTGTGAT 471 Hu.DMD.Exon53.20.021TGATACTAACCTTGGTTTCT 472 Hu.DMD.Exon53.20.022 ATCTTTGATACTAACCTTGG 473Hu.DMD.Exon53.20.023 AAGGTATCTTTGATACTAAC 474 Hu.DMD.Exon53.20.024TTAAAAAGGTATCTTTGATA 475 Hu.DMD.Exon54.25.001 CTATAGATTTTTATGAGAAAGAGA476 Hu.DMD.Exon54.25.002 AACTGCTATAGATTTTTATGAGAAA 477Hu.DMD.Exon54.25.003 TGGCCAACTGCTATAGATTTTTATG 478 Hu.DMD.Exon54.25.004GTCTTTGGCCAACTGCTATAGATTT 479 Hu.DMD.Exon54.25.005CGGAGGTCTTTGGCCAACTGCTATA 480 Hu.DMD.Exon54.25.006ACTGGCGGAGGTCTTTGGCCAACTG 481 Hu.DMD.Exon54.25.007TTTGTCTGCCACTGGCGGAGGTCTT 482 Hu.DMD.Exon54.25.008AGTCATTTGCCACATCTACATTTGT 483 Hu.DMD.Exon54.25.008.TTTGCCACATCTACATTTGTCTGCC 484 2 Hu.DMD.Exon54.25.009CCGGAGAAGTTTCAGGGCCAAGTCA 485 Hu.DMD.Exon54.25.010GTATCATCTGCAGAATAATCCCGGA 486 Hu.DMD.Exon54.25.010.TAATCCCGGAGAAGTTTCAGGGCCA 487 2 Hu.DMD.Exon54.25.011TTATCATGTGGACTTTTCTGGTATC 488 Hu.DMD.Exon54.25.012AGAGGCATTGATATTCTCTGTTATC 489 Hu.DMD.Exon54.25.012.ATGTGGACTTTTCTGGTATCATCTG 490 2 Hu.DMD.Exon54.25.013CTTTTATGAATGCTTCTCCAAGAGG 491 Hu.DMD.Exon54.25.013.ATATTCTCTGTTATCATGTGGACTT 492 2 Hu.DMD.Exon54.25.014CATACCTTTTATGAATGCTTCTCCA 493 Hu.DMD.Exon54.25.014.CTCCAAGAGGCATTGATATTCTCTG 494 2 Hu.DMD.Exon54.25.015TAATTCATACCTTTTATGAATGCTT 495 Hu.DMD.Exon54.25.015.CTTTTATGAATGCTTCTCCAAGAGG 496 2 Hu.DMD.Exon54.25.016TAATGTAATTCATACCTTTTATGAA 497 Hu.DMD.Exon54.25.017AGAAATAATGTAATTCATACCTTTT 498 Hu.DMD.Exon54.25.018GTTTTAGAAATAATGTAATTCATAC 499 Hu.DMD.Exon54.20.001 GATTTTTATGAGAAAGAGA500 Hu.DMD.Exon54.20.002 CTATAGATTTTTATGAGAAA 501 Hu.DMD.Exon54.20.003AACTGCTATAGATTTTTATG 502 Hu.DMD.Exon54.20.004 TGGCCAACTGCTATAGATTT 503Hu.DMD.Exon54.20.005  GTCTTTGGCCAACTGCTATA 504 Hu.DMD.Exon54.20.006CGGAGGTCTTTGGCCAACTG 505 Hu.DMD.Exon54.20.007 TTTGTCTGCCACTGGCGGAG 506Hu.DMD.Exon54.20.008 TTTGCCACATCTACATTTGT 507 Hu.DMD.Exon54.20.009TTCAGGGCCAAGTCATTTGC 508 Hu.DMD.Exon54.20.010 TAATCCCGGAGAAGTTTCAG 509Hu.DMD.Exon54.20.011 GTATCATCTGCAGAATAATC 510 Hu.DMD.Exon54.20.012ATGTGGACTTTTCTGGTATC 511 Hu.DMD.Exon54.20.013 ATATTCTCTGTTATCATGTG 512Hu.DMD.Exon54.20.014 CTCCAAGAGGCATTGATATT 513 Hu.DMD.Exon54.20.015CTTTTATGAATGCTTCTCCA 514 Hu.DMD.Exon54.20.016 CATACCTTTTATGAATGCTT 515Hu.DMD.Exon54.20.017 TAATTCATACCTTTTATGAA 516 Hu.DMD.Exon54.20.018TAATGTAATTCATACCTTTT 517 Hu.DMD.Exon54.20.019 AGAAATAATGTAATTCATAC 518Hu.DMD.Exon54.20.020 GTTTTAGAAATAATGTAATT 519 Hu.DMD.Exon55.25.001CTGCAAAGGACCAAATGTTCAGATG 520 Hu.DMD.Exon55.25.002TCACCCTGCAAAGGACCAAATGTTC 521 Hu.DMD.Exon55.25.003CTCACTCACCCTGCAAAGGACCAAA 522 Hu.DMD.Exon55.25.004TCTCGCTCACTCACCCTGCAAAGGA 523 Hu.DMD.Exon55.25.005CAGCCTCTCGCTCACTCACCCTGCA 524 Hu.DMD.Exon55.25.006CAAAGCAGCCTCTCGCTCACTCACC 525 Hu.DMD.Exon55.25.007TCTTCCAAAGCAGCCTCTCGCTCAC 526 Hu.DMD.Exon55.25.007.TCTATGAGTTTCTTCCAAAGCAGCC 527 2 Hu.DMD.Exon55.25.008GTTGCAGTAATCTATGAGTTTCTTC 528 Hu.DMD.Exon55.25.008.GAACTGTTGCAGTAATCTATGAGTT 529 2 Hu.DMD.Exon55.25.009TTCCAGGTCCAGGGGGAACTGTTGC 530 Hu.DMD.Exon55.25.010GTAAGCCAGGCAAGAAACTTTTCCA 531 Hu.DMD.Exon55.25.010.CCAGGCAAGAAACTTTTCCAGGTCC 532 2 Hu.DMD.Exon55.25.011TGGCAGTTGTTTCAGCTTCTGTAAG 533 Hu.DMD.Exon55.25.011.TTCAGCTTCTGTAAGCCAGGCAAGA 635 2 Hu.DMD.Exon55.25.012GGTAGCATCCTGTAGGACATTGGCA 534 Hu.DMD.Exon55.25.012.GACATTGGCAGTTGTTTCAGCTTCT 535 2 Hu.DMD.Exon55.25.013TCTAGGAGCCTTTCCTTACGGGTAG 536 Hu.DMD.Exon55.25.014CTTTTACTCCCTTGGAGTCTTCTAG 537 Hu.DMD.Exon55.25.014.GAGCCTTTCCTTACGGGTAGCATCC 538 2 Hu.DMD.Exon55.25.015TTGCCATTGTTTCATCAGCTCTTTT 539 Hu.DMD.Exon55.25.015.CTTGGAGTCTTCTAGGAGCCTTTCC 540 2 Hu.DMD.Exon55.25.016CTTACTTGCCATTGTTTCATCAGCT 541 Hu.DMD.Exon55.25.016.CAGCTCTTTTACTCCCTTGGAGTCT 542 2 Hu.DMD.Exon55.25.017CCTGACTTACTTGCCATTGTTTCAT 543 Hu.DMD.Exon55.25.018AAATGCCTGACTTACTTGCCATTGT 544 Hu.DMD.Exon55.25.019AGCGGAAATGCCTGACTTACTTGCC 545 Hu.DMD.Exon55.25.020GCTAAAGCGGAAATGCCTGACTTAC 546 Hu.DMD.Exon55.20.001 AAGGACCAAATGTTCAGATG547 Hu.DMD.Exon55.20.002 CTGCAAAGGACCAAATGTTC 548 Hu.DMD.Exon55.20.003TCACCCTGCAAAGGACCAAA 549 Hu.DMD.Exon55.20.004 CTCACTCACCCTGCAAAGGA 550Hu.DMD.Exon55.20.005 TCTCGCTCACTCACCCTGCA 551 Hu.DMD.Exon55.20.006CAGCCTCTCGCTCACTCACC 552 Hu.DMD.Exon55.20.007 CAAAGCAGCCTCTCGCTCAC 553Hu.DMD.Exon55.20.008 TCTATGAGTTTCTTCCAAAG 554 Hu.DMD.Exon55.20.009GAACTGTTGCAGTAATCTAT 555 Hu.DMD.Exon55.20.010 TTCCAGGTCCAGGGGGAACT 556Hu.DMD.Exon55.20.011 CCAGGCAAGAAACTTTTCCA 557 Hu.DMD.Exon55.20.012TTCAGCTTCTGTAAGCCAGG 558 Hu.DMD.Exon55.20.013 GACATTGGCAGTTGTTTCAG 559Hu.DMD.Exon55.20.014 GGTAGCATCCTGTAGGACAT 560 Hu.DMD.Exon55.20.015GAGCCTTTCCTTACGGGTAG 561 Hu.DMD.Exon55.20.016 CTTGGAGTCTTCTAGGAGCC 562Hu.DMD.Exon55.20.017 CAGCTCTTTTACTCCCTTGG 563 Hu.DMD.Exon55.20.018TTGCCATTGTTTCATCAGCT 564 Hu.DMD.Exon55.20.019 CTTACTTGCCATTGTTTCAT 565Hu.DMD.Exon55.20.020 CCTGACTTACTTGCCATTGT 566 Hu.DMD.Exon55.20.021AAATGCCTGACTTACTTGCC 567 Hu.DMD.Exon55.20.022 AGCGGAAATGCCTGACTTAC 568Hu.DMD.Exon55.20.023 GCTAAAGCGGAAATGCCTGA 569 H50A(+02+30)-AVI-5656CCACTCAGAGCTCAGATCTTCTAACTTC 584 C H50D(+07−18)-AVI-5915GGGATCCAGTATACTTACAGGCTCC 585 H50A(+07+33) CTTCCACTCAGAGCTCAGATCTTCTAA586 H51A(+61+90)-AVI-4657 ACATCAAGGAAGATGGCATTTCTAGTTT 587 GGH51A(+66+95)-AVI-4658 CTCCAACATCAAGGAAGATGGCATTTCT 588 AG H51A(+111+134)TTCTGTCCAAGCCCGGTTGAAATC 589 H51A(+175+195) CACCCACCATCACCCTCYGTG 590H51A(+199+220) ATCATCTCGTTGATATCCTCAA 591 H51A(+66+90)ACATCAAGGAAGATGGCATTTCTAG 592 H51A(−01+25) ACCAGAGTAACAGTCTGAGTAGGAGC593 h51AON1 TCAAGGAAGATGGCATTTCT 594 h51AON2 CCTCTGTGATTTTATAACTTGAT 595H51D(+08−17) ATCATTTTTTCTCATACCTTCTGCT 596 H51D(+16−07)CTCATACCTTCTGCTTGATGATC 597 hAON#23 TGGCATTTCTAGTTTGG 598 hAON#24CCAGAGCAGGTACCTCCAACATC 599 H44A(+61+84) TGTTCAGCTTCTGTTAGCCACTGA 600H44A(+85+104) TTTGTGTCTTTCTGAGAAAC 601 h44AON1 CGCCGCCATTTCTCAACAG 602H44A(−06+14) ATCTGTCAAATCGCCTGCAG 603 H45A(+71+90) TGTTTTTGAGGATTGCTGAA604 h45AON1 GCTGAATTATTTCTTCCCC 605 h45AON5 GCCCAATGCCATCCTGG 606H45A(−06+20) CCAATGCCATCCTGGAGTTCCTGTAA 607 H53A(+39+69)CATTCAACTGTTGCCTCCGGTTCTGAAG 608 GTG H53A(+23+47)CTGAAGGTGTTCTTGTACTTCATCC 609 h53AON1 CTGTTGCCTCCGGTTCTG 610H53A(−12+10) ATTCTTTCAACTAGAATAAAAG 611 huEx45.30.66GCCATCCTGGAGTTCCTGTAAGATACC 612 AAA huEx45.30.71CCAATGCCATCCTGGAGTTCCTGTAAG 613 ATA huEx45.30.79GCCGCTGCCCAATGCCATCCTGGAGTT 614 CCT huEx45.30.83GTTTGCCGCTGCCCAATGCCATCCTGG 615 AGT huEx45.30.88CAACAGTTTGCCGCTGCCCAATGCCAT 616 CCT huEx45.30.92CTGACAACAGTTTGCCGCTGCCCAATG 617 CCA huEx45.30.96TGTTCTGACAACAGTTTGCCGCTGCCC 618 AAT huEx45.30.99CAATGTTCTGACAACAGTTTGCCGCTG 619 CCC huEx45.30.103CATTCAATGTTCTGACAACAGTTTGCCG 620 CT huEx45.30.120TATTTCTTCCCCAGTTGCATTCAATGTT 621 CT huEx45.30.127GCTGAATTATTTCTTCCCCAGTTGCATT 622 CA huEx45.30.132GGATTGCTGAATTATTTCTTCCCCAGTT 623 GC huEx45.30.137TTTGAGGATTGCTGAATTATTTCTTCCC 624 CA huEx53.30.84GTACTTCATCCCACTGATTCTGAATTCT 625 TT huEx53.30.88TCTTGTACTTCATCCCACTGATTCTGAA 626 TT huEx53.30.91TGTTCTTGTACTTCATCCCACTGATTCT 627 GA huEx53.30.103CGGTTCTGAAGGTGTTCTTGTACTTCAT 628 CC huEx53.30.106CTCCGGTTCTGAAGGTGTTCTTGTACTT 629 CA huEx53.30.109TGCCTCCGGTTCTGAAGGTGTTCTTGTA 630 CT huEx53.30.112TGTTGCCTCCGGTTCTGAAGGTGTTCTT 631 GT huEx53.30.115AACTGTTGCCTCCGGTTCTGAAGGTGT 632 TCT huEx53.30.118TTCAACTGTTGCCTCCGGTTCTGAAGGT 633 GT h50AON1 h50AON2Peptide Transporters NH₂ to COOH)*: rTAT RRRQRRKKRC 570 R₉F₂RRRRRRRRRFFC 571 (RRAhx)₄B RRAhxRRAhxRRAhxRRAhxB 572(RAhxR)₄AhxB; (P007) RAhxRRAhxRRAhxRRAhxRAhxB 573 (AhxRR)₄AhxBAhxRRAhxRRAhxRRAhxRRAhxB 574 (RAhx)₆B RAhxRAhxRAhxRAhxRAhxRAhxB 575(RAhx)₈B RAhxRAhxRAhxRAhxRAhxRAhxRAhxRAhx 576 B (RAhxR)₅AhxBRAhxRRAhxRRAhxRRAhxRRAhxRAhxB 577 (RAhxRRBR)₂AhxB; RAhxRRBRRAhxRRBRAhxB578 (CPO6062) MSP ASSLNIA 579Cell Penetrating Peptide/Homing Peptide/PMO Conjugates(NH₂ to COOH and 5′ to 3′) MSP-PMO ASSLNIA-XB- 580GGCCAAACCTCGGCTTACCTGAAAT 636 CP06062-MSP-PMO RXRRBRRXRRBR-XB-ASSLNIA-X-581 GGCCAAACCTCGGCTTACCTGAAAT 636 MSP-CP06062-PMOASSLNIA-X-RXRRBRRXRRBR-B- 582 GGCCAAACCTCGGCTTACCTGAAAT 636 CP06062-PMORXRRBRRXRRBR-XB- 583 GGCCAAACCTCGGCTTACCTGAAAT 636 *Ahx is6-aminohexanoic acid and B is beta-alanine.

It is claimed:
 1. An antisense oligonucleotide of 21 bases comprising abase sequence that is 100% complementary to 21 consecutive bases of exon50 of the human dystrophin pre-mRNA, wherein the base sequence comprises17 consecutive bases of AGGCTCCAATAGTGGTCAGTCCAGG (SEQ ID NO:285), inwhich thymine bases are uracil bases, wherein the antisenseoligonucleotide is a 2′-O-methyl oligonucleotide, and wherein theantisense oligonucleotide induces exon 50 skipping; or apharmaceutically acceptable salt thereof.
 2. An antisenseoligonucleotide of 21 bases comprising a base sequence that is 100%complementary to 21 consecutive bases of exon 50 of the human dystrophinpre-mRNA, wherein the base sequence comprises 17 consecutive bases ofAGGCTCCAATAGTGGTCAGTCCAGG (SEQ ID NO:285), in which: (i) thymine basesare uracil bases and (ii) cytosine bases are 5-methylcytosine bases,wherein the antisense oligonucleotide is a 2′-O-methyl oligonucleotide,and wherein the antisense oligonucleotide induces exon 50 skipping; or apharmaceutically acceptable salt thereof.
 3. An antisenseoligonucleotide of 21 bases comprising a base sequence that is 100%complementary to 21 consecutive bases of exon 50 of the human dystrophinpre-mRNA, wherein the base sequence comprises 17 consecutive bases ofAGGCTCCAATAGTGGTCAGTCCAGG (SEQ ID NO:285), in which: (i) thymine basesare uracil bases and (ii) one or more of the bases are hypoxanthine,wherein the antisense oligonucleotide is a 2′-O-methyl oligonucleotide,and wherein the antisense oligonucleotide induces exon 50 skipping; or apharmaceutically acceptable salt thereof.
 4. An antisenseoligonucleotide of 21 bases comprising a base sequence that is 100%complementary to 21 consecutive bases of exon 50 of the human dystrophinpre-mRNA, wherein the base sequence comprises 17 consecutive bases ofAGGCTCCAATAGTGGTCAGTCCAGG (SEQ ID NO:285), in which: (i) thymine basesare uracil bases, (ii) one or more of the bases are hypoxanthine, and(iii) cytosine bases are 5-methylcytosine bases, wherein the antisenseoligonucleotide is a 2′-O-methyl oligonucleotide, and wherein theantisense oligonucleotide induces exon 50 skipping; or apharmaceutically acceptable salt thereof.
 5. A pharmaceuticalcomposition comprising an antisense oligonucleotide of 21 basescomprising a base sequence that is 100% complementary to 21 consecutivebases of exon 50 of the human dystrophin pre-mRNA, wherein the basesequence comprises 17 consecutive bases of AGGCTCCAATAGTGGTCAGTCCAGG(SEQ ID NO: 285), in which thymine bases are uracil bases, wherein theantisense oligonucleotide is a 2′-O- methyl oligonucleotide, and whereinthe antisense oligonucleotide induces exon 50 skipping; or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.
 6. A pharmaceutical composition comprising anantisense oligonucleotide of 21 bases comprising a base sequence that is100% complementary to 21 consecutive bases of exon 50 of the humandystrophin pre-mRNA, wherein the base sequence comprises 17 consecutivebases of AGGCTCCAATAGTGGTCAGTCCAGG (SEQ ID NO: 285), in which: (i)thymine bases are uracil bases and (ii) cytosine bases are5-methylcytosine bases, wherein the antisense oligonucleotide is a2′-O-methyl oligonucleotide, and wherein the antisense oligonucleotideinduces exon 50 skipping; or a pharmaceutically acceptable salt thereof,and a pharmaceutically acceptable carrier.
 7. A pharmaceuticalcomposition comprising an antisense oligonucleotide of 21 basescomprising a base sequence that is 100% complementary to 21 consecutivebases of exon 50 of the human dystrophin pre-mRNA, wherein the basesequence comprises 17 consecutive bases of AGGCTCCAATAGTGGTCAGTCCAGG(SEQ ID NO: 285), in which: (i) thymine bases are uracil bases and (ii)one or more of the bases are hypoxanthine, wherein the antisenseoligonucleotide is a 2′-O-methyl oligonucleotide, and wherein theantisense oligonucleotide induces exon 50 skipping; or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.
 8. A pharmaceutical composition comprising anantisense oligonucleotide of 21 bases comprising a base sequence that is100% complementary to 21 consecutive bases of exon 50 of the humandystrophin pre-mRNA, wherein the base sequence comprises 17 consecutivebases of AGGCTCCAATAGTGGTCAGTCCAGG (SEQ ID NO: 285), in which: (i)thymine bases are uracil bases, (ii) one or more of the bases arehypoxanthine, and (iii) cytosine bases are 5-methylcytosine bases,wherein the antisense oligonucleotide is a 2′-O-methyl oligonucleotide,and wherein the antisense oligonucleotide induces exon 50 skipping; or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.